Acute leucocyte, muscle damage, and stress marker responses to high-intensity functional training.

BACKGROUND: High-intensity functional training (HIFT) has become more popular, and the number of practitioners has increased; however, it remains unclear whether perturbations in the immune parameters occur, even after one single bout. Our aim was to examine acute leucocyte, muscle damage, and stress marker responses following a single 'Cindy' workout session, and compare the results between novice and experienced participants.
MATERIAL AND METHODS: Twenty-three HIFT practitioners (age 31.0 ± 1.0 years) completed the 'Cindy' workout. They were categorized as novice (3-8 months of experience; n = 10) and experienced (≥18 months; n = 13). White blood cell (WBC) count, plasma creatine kinase (CK) activity, blood cortisol level, and lactate concentration were measured. Blood analysis was performed before (pre-ex), immediately after (post-ex), 30 min after (post-30 min), and 24 h after (post-24 h) a single 'Cindy' workout session.
RESULTS: WBC count was higher post-ex (6.8 to 11.8x103/μL) and returned to baseline values within post-30 min (p<0.01). Neutrophil (3.3 to 4.5x103/μL) and lymphocyte levels (2.8 to 5.9x103/μL) were higher post-ex and returned to baseline values after post-24 h, yet lymphocytopoenia (2.2x103/μL) was observed at post-30 min (p<0.01). CK increased post-ex (174.9 to 226.7 U.L-1) and remained elevated post-24 h. Cortisol (14.7 to 17.0 μg/dL) and lactate (1.9 to 13.5 mmol.l-1) responses increased post-ex, but only the lactate level was reduced at post-30 min (p<0.01). The experienced participants had higher WBC, lymphocyte, and cortisol concentrations post-ex than the novice ones (p<0.01).
CONCLUSIONS: A single HIFT session elicited significant acute perturbations in WBC count, stress markers, and muscle tissue, which is like other similar regimens. Importantly, the experienced participants showed greater lymphocyte and cortisol responses than the novice ones.

Introduction

High-intensity functional training (HIFT) is a form of exercise enjoyed recreationally by participants of varying levels of fitness, training experience, age, and lifestyles, and it is also considered a sport on its own [1-3]. This exercise regimen characterized by high intensity, constant variation, and functional movement is often performed in rapid, successive repetition with limited or no recovery time [4-7]. HIFT is based on the concept of increased work capacity over time while using a variety of exercise modalities, including mono-structural (e.g. running, rowing, etc.), as well as body weight movements (e.g. squats, push-ups, etc.) and weightlifting derivatives (e.g. snatch, shoulder press, deadlift, etc.) [4, 6, 8].

HIFT mixes endurance, power, and strength within the same sport and exercise, at the same time, making it unlike any other. It may be the most comprehensive sport modality, as it requires all domains of physical conditioning [9]. HIFT programs are designed to improve parameters of general physical fitness (e.g. cardiovascular endurance, strength, body composition, flexibility, etc.) and performance (e.g. agility, speed, power, strength, etc.) [8, 10]. Nevertheless, some features of HIFT programs do not seem to follow the norms for safe and proper muscular fitness development, which leads to high health risks and an elevated risk of injury [7, 11–13].

The most popular among these HIFT regimens is CrossFit®, a short-duration, high-volume and high-intensity exercise program [3, 6]. Its training is organized as daily sessions called ‘workouts of the day’ (WODs) which usually take 20 min or less, and it is done with short or no rest periods, causing the participants to be susceptible to changes in immune parameters, endocrine activities, and redox status [5, 9, 14, 15]. The workouts in this program are based on three modalities: gymnastics, metabolic conditioning, and weightlifting [16].

Recently, investigators have demonstrated that a HIFT bout elicited significant increases in interleukin-6 (IL-6), IL-10, lactate, and glucose concentrations after WODs [5]. An acute blood oxidative stress response comparable to a traditional bout of high-intensity treadmill running in male HIFT practitioners was also observed [14]. Similarly, Heavens et al. [17] showed that a high-intensity, short-rest protocol (with characteristics similar to those of a HIFT session) elicits significant increases in indirect blood markers of muscle damage. As far as we know, only one study has chronically evaluated immunological responses in HIFT practitioners [13]. Despite the popularity of HIFT, very few studies have taken the impact of a single training session on physiological variables, such as metabolic markers, immune biomarkers, stress markers, redox status, and hormonal aspects into consideration [2, 5, 9, 14].

Therefore, we focused on outcomes, like immune parameters, during a 24-h recovery period. To the best of our knowledge, this is the first study to analyze the effects of a single HIFT session on parameters related to immune cell changes, such as white blood cell (WBC) count. Thus, the present study aimed (1) to examine the WBC count, plasma creatine kinase (CK) activity, cortisol concentration, and lactate response following a single ‘Cindy’ workout session, and (2) to compare the differences between novice and experienced participants. Specifically, the hypotheses tested were as follows: a single ‘Cindy’ workout session would induce perturbations in immune parameters after a training session, and novice participants would be more susceptible to these changes when compared to experienced participants.

Materials and methods

Experimental approach to the problem

This study was conducted over a total of five visits, comprehending anthropometric evaluation, familiarization with the WODs, and food record explanation (visit 1); one repetition maximum (1RM) test (visit 2); fitness test (visit 3); ‘Cindy’ workout (visit 4); and a blood sample collection 24h later (visit 5). Prior to and after the training session, blood samples were collected from the antecubital vein and transferred to vacuum tubes (Vacutainer; Becton Dickinson, USA) for further analysis of immune parameters, endocrine activities, and biochemical responses.

On the first visit, volunteers underwent anthropometric measurements (body mass, body height, and skinfold thicknesses) [18], familiarization with the ‘Cindy’ workout, and eating pattern assessment. For the food record, participants were asked to record three non-consecutive days of their food intake so that their energy and macronutrient intakes could be calculated later using the software “Dietpro” version 5.1. All participants were instructed on how to properly log their food, snacks, and drinks. They received illustrative material with explanations about portion sizes and dietary technical, and they were told not to consume caffeine-rich drinks or foods (e.g. tea, coffee, and chocolate), or alcoholic beverages for 24 h in order to avoid any interference with body hydration. On the second visit (after 48 h), the participants performed the deadlift exercise for 1RM test [19]. After a 24-h rest, on the third visit, the participants performed the yo-yo intermittent recovery test (yo-yo IR1) [20], during which they were monitored through a heart rate (HR) monitor (Polar Team Pro, Kempele, Finland), and the maximum HR was recorded. Strong verbal encouragement was provided to elicit maximal effort during both tests. All tests took place at the same CrossFit® gym. On visit 4, each test was carried out between 6h00 AM and 8h00 AM to avoid circadian cycle influences. Volunteers were asked to refrain from physical exercise for 48 h. Females were tested in the early follicular phase of the menstrual cycle (2–7 days after the onset of menses) to minimize the effect of hormonal fluctuations on outcome measures.

Subjects

Healthy volunteers were recruited from a CrossFit® affiliate gym (‘CrossFit® Quest’, Brazil). Inclusion criteria for participant selection were at least 3 months of experience in HIFT routines and the ability to perform the ‘Cindy’ workout. Novice (NOV) participants were defined as those with 3–8 months of experience, while experienced (EXP) participants were those who had more than 18 months of experience. These timelines were selected to ensure a significant gap in experience level between the two groups of participants [21]. Subjects were excluded if they presented (a) 9–17 months of experience; (b) any injury or motor impairment; (c) any cardiovascular, metabolic, or neurological diseases; (d) the use of any type of medication or performance-enhancing drugs; (e) the use of supplements containing antioxidant compounds in the last six weeks, as well as those considered ergogenic [22], such as caffeine, creatine, beta-alanine, nitrate, and bicarbonates in the last four months; (f) incomplete 'Cindy' workout on visit 4; (g) onset of symptoms of upper respiratory tract infections at any time during the experimental design; and (h) failure to consume the pre-workout standardized breakfast on visit 4.

Ethical approval was obtained from the local review board (Research Ethics Committee of Federal University of Sergipe, process no. 3.087.955/2018), and the complete project was registered in the Brazilian Clinical Trials Registry (ReBEC RBR-2GH23P). Participation was voluntary, and all participants signed an informed consent document before taking part in the study, which was conducted in accordance with the Declaration of Helsinki with the recent amendment of Fortaleza (2013). All the individuals selected for the study were considered able to perform the exercise based on Par-Q (Canada’s physical activity guide to healthy active living) [23]. Although experienced participants have had more than 18 months of experience, they were classified as recreational as they have never participated in an official competition (except those organized by the gym itself). Participants commonly perform three to five whole-body exercise sessions per week. To sample characterization, motor performance tests (deadlift and yo-yo test) and body composition evaluation were performed prior to the intervention. These evaluations are shown in the results section (Table 1).

Table 1

Characteristics of the participants (M ± SEM [SD]).

Variables	EXP (13)	NOV (10)	p-value	All (23)
Age (years)	31.1 ± 1.4 [4.9]	30.9 ± 1.5 [4.8]	0.932	31.0 ± 1.0 [4.8]
Sex	7 M 6 F	5 M 5 F	0.855	12 M 11 F
Experience (months)	28.5 ± 1.8 [6.4]	6.0 ± 0.5 [1.5]	0.001	18.7 ± 2.5 [12.4]
Weight (kg)	70.8 ± 3.7 [13.3]	78.5 ± 4.5 [14.1]	0.197	74.2 ± 2.9 [13.9]
Height (cm)	1.69 ± 0.03 [0.10]	1.69 ± 0.03 [0.09]	0.908	1.69 ± 0.02 [0.09]
BMI (kg/m2)	24.4 ± 0.7 [2.5]	27.3 ± 0.8 [2.7]	0.017	25.7 ± 0.6 [2.9]
Body Fat (%)	17.4 ± 1.4 [5.1]	22.5 ± 1.7 [5.4]	0.031	19.6 ± 1.2 [5.7]
Deadlift (kg)	131.6 ± 11.5 [41.6]	105.2 ± 8.7 [27.6]	0.098	120.6 ± 7.9 [37.9]
Yoyo RL1 (m)	507.7 ± 57.9 [208.7]	336.0 ± 52.4 [165.7]	0.045	424.3 ± 43.0 [206.3]
VO2max (ml/kg/min)	40.7 ± 0.5 [1.8]	39.2 ± 0.4 [1.4]	0.045	40.0 ± 0.4 [1.7]
HRmax (bpm)	183.2 ± 2.9 [10.3]	189.4 ± 2.1 [6.6]	0.111	185.9 ± 1.7 [9.3]

M = mean; SEM = standard error of the mean; SD = standard deviation.

M = male; F = female; BMI = body mass index; Yoyo RL1 = yoyo recovery test level 1; VO2max = maximum oxygen consumption; HRmax = heart rate maximum; EXP = experienced group; NOV = novice group.

Intervention

On visit 4, after a 48-h rest interval from visit 3, volunteers reported to the gym in the morning. The HIFT session performed was the gymnastics WOD called the ‘Cindy’ workout [16, 24]. The session started with a warm-up consisting of 5 min of low-intensity running and 5 min of joint mobility and dynamic stretching exercises. This WOD consisted of as many rounds as possible of 5 pull-ups, 10 push-ups, and 15 air squats in 20 min. The assistants of the authors were in charge of counting the rounds. The techniques used for each exercise have been described in detail in the literature [24]. When necessary, small modifications of the exercises (e.g. push-up on knees and ring rows for some women and men with insufficient strength) were made [25]. The ‘Cindy’ workout was chosen because it caused the greatest metabolic and cardiovascular stress when compared to other protocols [14, 15, 26]. Before the start of the study, all volunteers had a familiarization session (visit 1), providing them with technical lessons on how to do the exercises. Ambient conditions for session were as follows: temperature of 25–29°C and relative humidity of 55–65%.

During the ‘Cindy’ workout, the subjects were monitored using an HR monitor (Polar Team Pro, Kempele, Finland). HR data were stored and subsequently extracted using the Polar Team 2 Pro system. Ratings of perceived exertion (REP), whose use was strongly recommended in a HIFT metabolic training session [6, 7, 27] were obtained using the CR10 Borg RPE scale [28]. The subjects were asked this question: ‘how hard do you feel the exercise was?’. RPE measurements were taken 30 min after the ‘Cindy’ workout. One hour before the training session, the volunteers consumed a standardized breakfast (approximately 320–350 cal) with a protein to fat to carbohydrate ratio of 20-35-45 (protein, fat, carbohydrate as percentage). These percentages culminated in the intake of approximately 40g carbohydrates, 17.5g protein, and 13g fat.

Blood collection

Before (pre-ex), immediately after (post-ex), 30 min afer (post-30 min), and 24 h after (post-24 h) exercise, blood samples (~8 mL) were obtained from the antecubital vein and transferred to vacuum tubes. The blood was analyzed for leucocyte (WBC), neutrophil, lymphocyte, monocyte (tubes mixed with ethylenediamine tetraacetic acid (EDTA)), and CK concentrations at pre-ex, post-ex, post-30 min, and post-24 h. Cortisol (tubes with serum gel) and blood lactate concentrations (tubes with sodium fluoride) were obtained at pre-ex, post-ex, and post-30 min (Fig 1). Leucocyte, neutrophil, lymphocyte, and monocyte were analyzed using an automatic haematology analyzer (Cell-Dyn Ruby System; Abbott Laboratories, Abbott Park, IL, USA). CK and lactate were measured with commercial kits, according to the manufacturer’s recommendations and using an automatic biochemistry analyzer (CMD 800i, Wiener Lab., Rosario, Argentina). Cortisol was measured with a commercial kit, according to the manufacturer’s recommendations and using an immunoassay analyzer (Abbott ARCHITECT i1000SR, Abbott Park, IL, USA).

Fig 1

Time points of blood collection.

PRE-EX = before exercise; POST-EX = immediately after exercise; POST-30min = post 30 minutes after exercise; POST-24h = 24h after exercise. HR = heart rate; CK = creatine kinase concentrations.

Statistical analyses

The normality and homogeneity of the variances were verified using the Shapiro–Wilk and Levene tests, respectively. Data are presented as mean and standard error of the estimate of the mean. In order to compare the mean values of the descriptive variables between the groups (EXP vs. NOV), an independent and a paired t-test were used. A 2 × 4 repeated measures analysis of variance (ANOVA) (interaction groups [EXP vs. NOV] × time [moments]) was used to compare the blood analysis. Post hoc comparisons were performed using the Bonferroni test (with correction). Assumptions of sphericity were evaluated using the Mauchly’s test. Where sphericity was violated (p<0.05), the Greenhouse–Geisser correction factor was applied. The Spearman product moment correlation was used to examine the relationship between rounds and immune system, and hormonal parameters. The magnitude of the correlation coefficients was considered weak (0.10.7). Nonparametric statistics were used for the following variables: age, time, experience, and lymphocyte level at pre-ex, at post-30 min, and at post-24 h, and CK activity at post-24 h. All analyses were conducted using SPSS-22.0 software (IBM, SPSS Inc., Chicago, IL, USA). Significance was set at p<0.05.

Results

Out of 224 volunteers, 118 met the inclusion criteria (minimum of 3 months of experience and the ability to perform the ‘Cindy’ workout). The reasons for the other volunteers being excluded from the study were as follows: 82 for consuming supplements described in the exclusion criteria; three for having 9–17 months of experience; three for having metabolic diseases; three for not completing the workout; two for having a cold; and two for not consuming the standardized breakfast. After the selection process, 23 participants completed the study.

Neither groups (EXP or NOV) differed in terms of age, sex, height, weight, deadlift 1RM test, and HRmax in yo-yo IR1; nevertheless, BMI and body fat were lower, and VO2max and time experience were higher in the EXP group (Table 1).

Table 2 presents the mean number of rounds, cardiovascular variables, rate of perceived exertion, and RPE-number of rounds ratio for all participants and both groups. The mean number of rounds was higher, and RPE-number of rounds ratio was lower in the EXP in comparision with the NOV group, but HRmax, %HRmax, and RPE at post-30 min did not reveal any significant differences between the groups.

Table 2

Physiological and perceptual responses under the “Cindy” workout (M ± SEM [SD]).

Variables	EXP (13)	NOV (10)	p-value	All (23)
Number of rounds	15.1 ± 1.0 [3.5]	10.9 ± 0.8 [2.4]	0.004	13.3 ± 0.8 [3.7]
HRav (bpm)	173.4 ± 2.6 [9.5]	170.1 ± 2.3 [7.3]	0.377	172.0 ± 1.8 [8.6]
%HRmax	93.1 ± 0.6 [2.2]	93.0 ± 0.8 [2.4]	0.855	93.1 ± 0.5 [2.2]
RPE (post-30min)	7.5 ± 0.3 [1.2]	8.1 ± 0.3 [1.1]	0.262	7.8 ± 0.2 [1.2]
RPE/Number of rounds ratio	0.52 ± 0.3 [0.13]	0.77 ± 0.6 [0.21]	0.002	0.63 ± 0.4 [0.21]

M = mean; SEM = standard error of the mean; SD = standard deviation.

HRav = average heart rate; %HRmax = percentage of maximum heart rate; RPE = rate of perceived exertion; EXP = experienced group; NOV = novice group.

Leucocyte count

WBC level increased significantly following the ‘Cindy’ workout immediately post-ex compared to baseline values for all participants, returning to PRE values within post-30 min (p<0.01). There were no differences between the groups in WBC count (Fig 2).

Fig 2

Timeline of leucocytes (A), neutrophils (B), lymphocytes (C), and monocytes (D), corresponding to before (pre-ex), immediately after (post-ex), 30 minutes after (post-30min), and 24h after (post-24h) exercise. WBC = white blood cell; EXP = experienced group; NOV = novice group. *Significantly different from pre-ex (p < 0.05); ‡Significantly greater than post-30min and post-24h (p < 0.05); ≠Significantly different from NOV group (p < 0.05); $Significantly lower than post-ex only for NOV (p < 0.05); αSignificantly greater than post-30min (p < 0.05). P1 = time effect; P2 = group effect; P3 = interaction group x time.

There was a significant increase in neutrophil count post-ex and at post-30 min, returning to pre-ex values at 24 h after the end of the protocol for all participants (p<0.01). There were no differences between the groups for neutrophil count; however, only for the NOV group, post-30 min values were significantly decreased (p = 0.020) when compared to the values immediately post-ex (Fig 2).

Lymphocyte levels increased significantly post-ex and decreased significantly at post-30 min, returning to pre-ex values 24 h after the end of the protocol for all participants (p<0.01). Lymphocyte levels in the EXP were significantly higher (p = 0.005) post-ex than those in NOV subjects (Fig 2).

There was a significant increase in monocyte levels post-ex, which returned to pre-ex values within post-30 min and increased again 24 h after the end the protocol for all participants (p<0.01). Monocyte levels did not differ between the groups (Fig 2).

Table 3 presents the neutrophil-lymphocyte ratio (NLR) following a single ‘Cindy’ workout session. The NLR decreased significantly post-ex compared to baseline values, rising to values above those pre-ex and post-ex (p<0.01), and returning to pre-ex values at 24 h after the end of the protocol for all participants. There were no differences between the groups regarding the NLR. Neutrophil-cortisol ratio (NCR) increased significantly post-ex and decreased within post-30 min for all participants (p<0.01). NCR in the NOV was significantly higher post-ex (p = 0.021) than it was in the EXP participants.

Table 3

Pre- to post-changes in Neutrophil-Lymphocyte ratio and Neutrophil-Cortisol ratio following a single ‘Cindy’ workout session (M ± SEM [SD]).

Variables	Group	Pre-ex	Post-ex	Post-30 min	Post-24 h
NLR	EXP	1.20 ± 0.14 [0.50]	0.72 ± 0.07 [0.25] *‡	2.04 ± 0.22 [0.78]*	1.28 ± 0.18 [0.65]
	NOV	1.34 ± 0.10 [0.33]	0.91 ± 0.07 [0.22] *‡	1.58 ± 0.12 [0.39]	1.46 ± 0.16 [0.52]
	All	1.26 ± 0.09 [0.43]	0.80 ± 0.05 [0.25]*‡	1.84 ± 0.14 [0.67]&	1.36 ± 0.12 [0.59]
NCR	EXP	0.22 ± 0.02 [0.06]	0.24 ± 0.02 [0.08]≠	0.22 ± 0.02 [0.07]	-
	NOV	0.25 ± 0.02 [0.07]	0.34 ± 0.03 [0.11]$	0.24 ± 0.03 [0.09]	-
	All	0.23 ± 0.01 [0.07]	0.28 ± 0.02 [0.10]*α	0.23 ± 0.02 [0.08]	-

M = mean; SEM = standard error of the mean; SD = standard deviation.

NLR = Neutrophil/Lymphocyte ratio; NCR: Neutrophil/Cortisol ratio; Pre-ex = before exercise; Post-ex = immediately after exercise; Post-30min = 30 minutes after exercise; Post-24h = 24 hours after exercise; EXP = experienced group; NOV = novice group.

*Significantly different from pre-ex (p<0.01)

‡Significantly different from post-30min and post-24h (p<0.01)

&Significantly different from pre-ex, post-ex, and post-24h (p<0.01)

αSignificantly different from post-30min (p<0.01).

≠Significantly different from NOV (p<0.01)

$Significantly different from pre-ex and post-30min only for NOV (p<0.01)

Neutrophil/Lymphocyte ratio = Time effect: 0.000; Group effect: 0.958; Interaction group x time: 0.371.

Neutrophil/Cortisol ratio = Time effect: 0.557; Group effect: 0.001; Interaction group x time: 0.127.

Only leucocytes (r = 0.43; p = 0.040) and lymphocytes (r = 0.55; p = 0.006) post-ex showed moderate correlations with the number of rounds.

Muscle damage

Compared to pre-ex values, there were significant increases in CK at all time points for all participants. There were no differences between immediately after, 30 min after, and 24 h after exercise time points (p>0.01). There were no differences between the groups for CK (Fig 3).

Fig 3

Timeline of creatine kinase corresponding to before (pre-ex), immediately after (post-ex), 30 minutes after (post-30min), and 24h after (post-24h) exercise.

EXP = experienced group; NOV = novice group. *Significantly different from pre-ex (p < 0.05). P1 = time effect; P2 = group effect; P3 = interaction group x time.

Stress markers

As shown in Fig 4, lactate increased significantly post-ex and decreased within post-30 min, but values remained above baseline for all participants (p<0.01). There were no differences between the EXP and NOV individuals for this stress marker.

Fig 4

Timeline of lactate (A), cortisol (B) and area under the curve (AUC) of cortisol (C), corresponding to before (pre-ex), immediately after (post-ex) and post 30 minutes (post-30min) exercise. EXP = experienced group; NOV = novice group. *Significantly different from pre-ex (p < 0.05); &Significantly greater than pre-ex only for ALL and EXP (p < 0.05); ≠Significantly different between EXP and NOV group (p < 0.05); αSignificantly greater than post-30min (p < 0.05). P1 = time effect; P2 = group effect; P3 = interaction group x time.

There were significant increases in cortisol levels post-ex and at post-30 min for all participants (p<0.01). Cortisol concentration in the NOV participants was significantly higher (p = 0.045) than pre-ex values only at post-30 min. The cortisol concentration in EXP subjects was significantly higher both post-ex (p = 0.001) and at post-30 min (p = 0.036) than it was in the NOV ones. The area under the curve (AUC) of cortisol was also significantly higher in the EXP (p = 0.004) than in the NOV participants (Fig 4). Cortisol post-ex showed moderate correlations (r = 0.53; p = 0.010) with the numbers of rounds.

Dietary analysis

Table 4 shows the daily intake of energy and macronutrients during 3 non-consecutive days. Neither groups (EXP or NOV) presented any significant differences.

Table 4

Daily intake of energy and macronutrients during 3 non-consecutive days (M ± SEM [SD]).

Variables	EXP (9)	NOV (3)	p-value	All (12)
Energy (Kcal)	1744.0 ± 282.7 [848.0]	1175.5 ± 321.4 [556.7]	0.311	1601.9 ± 231.9 [803.4]
Energy/Kg (Kcal)	23.5 ± 3.8 [11.3]	15.5 ± 3.6 [6.2]	0.276	21.5 ± 3.1 [10.6]
CH (g)	203.6 ± 43.1 [129.2]	133.4 ± 52.9 [91.6]	0.410	186.1 ± 35.0 [121.1]
CH/Kg BM	3.0 ± 0.5 [1.6]	1.7 ± 0.6 [1.1]	0.253	2.7 ± 0.5 [1.6]
PTN (g)	93.2 ± 14.4 [43.1]	53.7 ± 14.4 [25.0]	0.171	83.3 ± 12.2 [42.3]
PTN/Kg BM	1.4 ± 0.2 [0.5]	0.7 ± 0.2 [0.3]	0.060	1.2 ± 0.2 [0.5]
Fat (g)	61.1 ± 6.5 [19.4]	49.2 ± 9.6 [16.6]	0.367	58.1 ± 5.4 [18.8]

M = mean; SEM = standard error of the mean; SD = standard deviation.

BM: Body mass; CH: Carbohydrate; PTN: Protein. EXP = experienced group; NOV = novice group.

Discussion

The present study investigated whether WBC count, plasma CK activity, cortisol concentration, and lactate response are affected by an acute HIFT session. The main findings of this study were: a) after a single ‘Cindy’ workout session, alterations in the number of circulating leucocytes and subsets occurred, varying slightly according to the level of training experience; b) the workout session also elicited significant increases in muscle damage; c) cortisol and lactate levels increased after the training session. To the best of our knowledge, this is the first study to analyze the acute effects of a HIFT session–gymnastics modality–on immune parameters and hormonal concentrations, and to provide fundamental information on these biomarkers. The results can be useful because of the increased number of HIFT practitioners, who may experience physiological changes in the quality and number of WBCs, thus affecting their capacity to resist common infections [29].

Unlike other studies, the participants in the present study have never engaged in competitions [5] and showed a lower level of physical fitness [15, 26], as observed by the minimum number of rounds (6 to 10 more rounds) and VO2max. However, in these studies, only men and younger adults were included as participants.

Assessing the average HR achieved during the ‘Cindy’ workout, we obtained a mean value of 172.0 ± 8.6 bpm (93.1 ± 2.3%HRmax). According to the American College of Sports Medicine (ACSM) guidelines, this %HRmax could be described as being close to that corresponding to maximum-intensity exercise [30]. Studies that used the ‘Cindy’ workout in their intervention also found high cardiovascular demand [15, 21, 31]. Therefore, we can consider that our initial intervention proposal through a HIFT session was achieved. The EXP and NOV groups had similar relative cardiovascular intensities, with 93.1 and 93.0%HRmax, respectively.

One of the findings of this study was the high work intensity reflected by blood lactate levels. The results reported in the present study are in agreement with other investigations that observed significantly elevated levels of lactate for the ‘Cindy’ workout in which this variable always exceeded 10 mmol/L-1 [14, 15, 26]. In our results, even after 30 min recovery, blood lactate levels remained above baseline (>2.8 mmol/L-1). There were no differences in lactate concentrations between the groups at any time point. In the same way, another intensity variable, such as RPE, had similar responses. RPE showed a moderate correlation with blood lactate 30 min after the session in a recent publication [7]. Comparable to lactate concentrations, there were no differences between the groups for RPE. On the other hand, when analyzing the RPE/Number of rounds ratio, we found disparities between the groups, showing that the EXP subjects perceive less effort per round.

It is believed that, even with a larger number of rounds (20 pull-ups, 40 push-ups, and 60 more air squats for the EXP group), the EXP participants showed better recovery capacity between rounds. The EXP group showed superior aerobic capacity (>1.5 mL/kg/min) than the NOV group, a fact also observed in a recently published study, in which superiority in aerobic capacity and HIFT performance was demonstrated in participants with more experience [32]. Higher aerobic capacity has been related to high lactate removal rate, since aerobic training can promote higher synthesis of monocarboxylate transporters [33].

However, despite this evidence, it is not possible to state that the largest VO2max of the EXP group is responsible for the difference in the number of rounds. Previous study evaluated practitioners who competed in the 2014 CrossFit Open and/or Regional competitions and concluded that, in this population studied, which performed an average of 23.3 rounds in ‘Cindy’ workout, VO2max cannot predict performance in the CrossFit® benchmark WOD [34]. It is worth mentioning that maximum strength level revealed no differences between groups. Although the NOV group had only 3 to 8 months of experience with HIFT, they were all well versed in strength training. Therefore, it is possible to speculate that the difference between EXP and NOV is explained by local muscle endurance, a parameter not evaluated in the present study.

In the present investigation, the cortisol concentrations increased immediately after exercise and remained elevated at post-30 min. Following extremely stressful exercise, the release of hormones, including testosterone, oestrogen, and cortisol is likely to occur [35, 36], and the highest cortisol increases immediately after exercise have been observed in high-intensity exercise protocols [17, 37]. When comaparing the groups, the EXP one showed a cortisol concentration significantly higher than that of the NOV group immediately after and 30 min after exercise, including when analyzed for the AUC. This may once again be explained by the fact that the higher number of rounds performed by the EXP group might simply be a reflection of the level of training presented by the NOV group. It is worth noting that a moderate correlation between cortisol and the number of rounds at the post-ex was found. This excess circulating cortisol in the body following high-intensity exercise protocols (e.g. the ‘Cindy’ workout) warns of some negative health outcomes, such as the saturation of many target receptors in the repair process and in the immune response [37].

After the ‘Cindy’ workout, there were alterations in the number of circulating leucocytes and subsets, which varied slightly according to the level of training experience. The total number of leucocytes circulating in peripheral blood is strongly influenced by physical exercise [29, 35]. In the present study, we found a significant increase in leucocyte counts post-ex. It has been attributed to the sympathetic activation and immediate release of catecholamines during exercise, promoting the demargination of leucocytes adhered to the marginal pool, as well as cellular recruitment from storage and synthesis tissues for circulation by mechanical action [35]. In the group comparison, the EXP group showed significantly higher lymphocyte counts post-ex than did the NOV one. Additionally, the higher lymphocyte counts may be related to the ability these participants have to recover more quickly for new sessions, as their immune system has a better memory of this type of stress [38, 39]. It is important to note that moderate correlations between the number of rounds with leukocytes and lymphocytes in the post-ex were also found.

Although WBC returned to pre-ex values within post-30 min, neutrophils remained above and lymphocytes below baseline values. We believe that the neutrophils and lymphocytes results at post-30 min are a consequence of the late action of cortisol, possibly mediated by IL-6. In maximum-intensity exercise, there is an increase in several cytokines in the blood circulation, IL-6 being the main one. Generally, IL-6 comes from the skeletal muscle and from the adipose tissue. When muscle damage occurs, immune cells infiltrate in the skeletal muscle and secrete IL-6 as well, causing an elevation of weak magnitude in the plasma concentration, but just in the period necessary to repair the damaged tissue. Significant increase in IL-6 in maximal exercise is mainly due to greater release by skeletal muscle [40]. In addition, chronic low carbohydrate intake and low muscle glycogen stores can stimulate IL-6 synthesis in exercise. IL-6 elevation in exercise culminates in a series of metabolic events, such as hepatic glycogenolysis and gluconeogenesis, fat degradation in adipocytes, muscle protein and amino acid degradation, and increased release of cortisol by the adrenal glands [41]. Increased cortisol has late effects, such as inhibition of mitogenesis and/or acceleration of lymphocyte apoptosis resulting in lymphocytopoenia, besides raising the mobilization of bone marrow neutrophils to blood, which increases the count of these cells at the end of the exercise sessions [35]. After maximum workout, lymphocytopoenia seems to have resulted from either death via apoptosis [42] or immune cell migration [43], or it may have been triggered by both [44].

In the present study, we believe that lymphocytopenia and the maintenance of high levels of neutrophils observed in the EXP group at post-30 min are a consequence of the late action of cortisol, probably in conjunction with IL-6, even though cytokine/myokine was not analyzed in the present study. Considering the NLR normality values (0.78–3.58) previously proposed [45], in the present study, although the NLR was within the normal range at all time points for both groups, at post-30 min it was observed to be higher than pre-ex only for the EXP group (more exposed to cortisol), corroborating the hypothesis that cortisol promotes a pro-neutrophil, anti-lymphocyte environment. Moreover, there was no difference between the NCR pre-ex and at post-30 min, which suggests the possible late action of cortisol on these white cells. It is worth mentioning that NCR at the post-ex does not justify the increase in neutrophils observed in the study, as expected, because in post-ex the catecholamines effects are usually more relevant.

The monocyte numbers quickly reverted to their original pre-ex values at post-30 min, and they showed delayed monocytosis at 24 h post-ex, as predicted. There is some evidence of increased monocyte count following 1.5–2.0 h of recovery after single bouts of endurance-based exercise [29, 35]. It is important to notice that none of the participants presented values above the reference for leucocyte indices, which is fundamental for optimal physical performance.

Plasma CK activity was used to assess muscle damage. CK is an intramuscular protein usually impermeable to the membrane which is unable to pass through it in its entirety. When damage occurs, there is fragility and rupture of the plasma membrane, and CK is released in the circulation and cleared from the blood by the reticuloendothelial system [17], which justifies the delayed release of CK into circulation.

A recent study evaluated the CK concentrations of 12 experienced male HIFT practitioners in two different types of WODs [46]. There was a significant increase immediately after exercise (WOD1 492 and WOD2 689 U/L-1), and the CK peak was observed 24 h after the intervention (WOD1 673 and WOD2 864 U/L-1), just as it occured in the present study. The initial CK level was 406 U/L-1 in WOD1 and 566 U/L-1 in WOD2. Another study followed nine participants for three days of HIFT competition [47]. The results showed that CK activity increased by 50% 24 h after the competition (472 U/L-1 at baseline and 698 U/L-1 at post-24 h). It is noteworthy that in the investigation of Tibana et al. (2019), the participants were exposed to exhaustive exercises for 3 consecutive days, whereas in our study all participants were exposed to 20 min of intervention. Additionally, the CK values presented by the referred study at pre-competition were higher than normal, demonstrating that, in this study, the participants had probably started the experiment without full recovery, similar to the results found by Timón et al. (2019).

With regard to the initial level (pre-ex) of CK, it is important to mention that, in our study, the participants had two days of absolute rest before the ‘Cindy’ workout, and it is probably for that reason that the initial levels of CK found in the other two studies [46, 47] were much higher than those in our findings (174 U/L-1 pre-ex). As for post-ex (immediately after), in the study by Timón et al. (2019) there was a significant CK increase when compared to pre-ex, just like observed in our study. All the studies [46, 47]–ours included–had significant CK elevations at post-24h compared to pre-ex, as expected. With regard to the increase in CK between post-ex and post-24h, it is possible to compare our study with that of Timón et al. (2019), which adopted percentage difference (Δ) values to analyze these time points. For that, we calculated Δ values of our study and found an increase of 22%, while Timón et al. (2019), in WOD2, observed an increase of 37% in the same interval time. The reason for our percentage difference not to be such high in CK between post-ex and post-24h, when compared to study mentioned above, may be the training pattern rather than its intensity [48], as simple and more commonly performed movements were adopted in the present study (pull-ups, push-ups and air squats), while more complex moviments (WOD 2: wall ball and power clean) were performed in the other study. Comparing the absolute values observed in the other studies [46, 47] with those obtained in the present study becomes impracticable, as the initial levels were already very different.

Special concerns, such as rhabdomyolysis, must be taken into consideration, as high-intensity, short-rest protocols could induce excessive muscle damage in athletes [37]. Rhabdomyolysis is a condition in which the excessive amount of damaged muscle tissue breaks down its intracellular contents, and abnormal CK (10.000–20.000 U/L) and myoglobin levels in relation to those expected for responses to exercise are released into the circulation, leading to secondary clinical and biochemical complications [49, 50]. Such condition did not occur in our study, considering that the highest average CK concentration at post-24 h was 276 U/L, with one subject with a 765 U/L maximum value of CK.

The EXP group performed significantly more work, leading to greater potential for structural damage; however, no differences in CK concentration between the groups were found at any time point. The similarity in CK increase in both groups, even with the EXP having done more work, can be explained by the fact that this phenomenon does not depend on the amount of work itself, but on the amount of work performed in comparison with the amount of work performed previously in preceding trainings. EXP practitioners performed more work because they were used to performing more work. Hence, the proportion between work performed in the present analysis and the one usually performed prior to it was possibly similar between the EXP and NOV groups. Another likely explanation is the major differences in CK response among our volunteers, just as observed in a recent investigation [47].

Nutrient intake has a relevant impact on the parameters analyzed in the present study. There were no differences between the groups with regard to energy and macronutrient intake; however, it is important to highlight that the absence of differences may also have occurred due to the reduced number of participants in the NOV group. The dietary pattern of the participants in the present study revealed insufficient intake of energy (<50 kcal/kg/day [22]) and carbohydrates (<5g/kg/day [22]). The energy and macronutrient intake found in the present study was lower than that observed in Overtraining Syndrome-affected HIFT athletes assessed in a recently published study [9]. It is possible that the participants of the present study have lower values than those of Cadegiani et al. (2019) due to ergogenic supplement intake (e.g. creatine), thus being considered an exclusion criterion. In general, creatine consumers also ingest hypercaloric supplements or proteins because such products claim to lead to muscle hypertrophy [22]. Low carbohydrate intake can promote immunological disorders and a higher rate of proteolysis (CK elevation), probably due to mechanisms that involve greater release of IL-6 from the skeletal muscle into the blood circulation during exercise [51]. Still, it is relevant to emphasize that at pre-ex, the volunteers consumed a standardized breakfast, with adequate amounts of carbohydrates [52], which would minimize the impact of nutrition on the findings of the present study.

In this study, some limitations should be highlighted, such as: a) the absence of inflammation marker measures which would allow us to make further inferences about the immune response; b) absence of lactate measure at 24h-post for the evaluation of the potential differences in lactate clearance speed between the groups, as a marker of speed recovery; c) absence of speed control or duration of each round; d) use of indirect test to assess VO2max, which may have underestimated the aerobic capacity of the participants; e) low adherence to the food record, even with a number lower than the one determined by the sample calculation to detect differences between the groups; f) focus limited on HIFT exercise that has a high metabolic demand, despite knowing that a traditional HIFT session is comprised of specific exercises for the development of strength, power, and gymnastics, which are usually performed before the WOD.

Several aspects demonstrate the need for further studies. In view of the acute effects of a HIFT session on immune response, muscle damage, and stress markers, additional research for investigating other health outcomes should be conducted with both experienced and novice practitioners. In addition, the safety of HIFT should be better understood and adapted to people with low physical fitness. During the ‘Cindy’ workout, one man and two women in the NOV group reported discomfort and dizziness in the final minutes of the session, not being able to complete the workout. Therefore, we believe that if the way the HIFT WODs is adjusted for performance, they could be applied to anyone, regardless of experience and conditioning level. A possible strategy in exercise prescription could be either a longer recovery time during the session or a decreased total training time, with gradual progression over the training weeks.

Conclusion

One single HIFT session elicited significant acute perturbations in WBC count, stress markers, and muscle tissue, similarly to other high-intensity training regimens of the kind. It is noteworthy that the EXP participants showed greater lymphocyte and cortisol responses than the NOV ones did.

Table 5.

Pre- to post-changes in outcomes of leucocytes, neutrophils, lymphocytes, monocytes, creatine kinase, lactate, cortisol and area under the curve of cortisol following a single ‘Cindy’ workout session (M ± SEM [SD]) *Significantly different from pre-ex (p < 0.05); ‡Significantly greater than post-30min and post-24h (p < 0.05); ≠Significantly different between EXP and NOV group (p < 0.05); $Significantly lower than post-ex only for NOV (p < 0.05); αSignificantly greater than post-30min (p < 0.05); &Significantly greater than pre-ex only for ALL and EXP (p < 0.05).

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7 Aug 2020

PONE-D-20-16542

Acute leucocyte, muscle damage, and stress marker responses to functional fitness programs Immune response to functional fitness programs

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Reviewer #1: General comments:

1. This is a simple yet well thought study. Authors found a gap on a very basic knowledge and explored it appropriately. These are those kind of basic studies that are more than necessary to better characterize the effects of this specific sport, with interesting peculiarities.

2. “Functional Fitness” has also been largely used as “High Intensity Functional Training” HIFT). Since its main representative is the branded sport “CrossFit”, it was kind of challenging to uniformize a generic name. I recommend authors to change for “HIFT” throughout the manuscript.

3. Authors should be careful when using the brand name (CrossFit). Its previous owners of the brand (the brand CrossFit has been recently sold) were threatening several research groups that attempted to describe scientific reports of CrossFit, and copiously have attempted to retract studies on “CrossFit”.

Introduction

Overall comment:

I suggest a brief explanation on why FFT/HIFT may elicit different effects than those of other sports, by comparing their characteristics, which the most fundamental ones are:

1. FFT/HIFT mixes endurance, explosion, and strength within the same sport, exercise, at the same time, unlike any other sport, and may be the only activity that requires all domains of physical conditioning.

2. There are not patterns or predictable sequence of exercises as well as the characteristics of exercise execution. This leads to higher caloric expenditure since It does not allow muscles to learn patterns and “predict” the following demand patterns. Hence, organism is constantly getting adapted. Perhaps, FFT/HIFT practitioners may develop a sort of an ability to “condition faster in conditioning processes”, i.e., their bodies develop specific conditioning abilities faster than any other sport, as they are constantly submitted to “unexpected” new physical demands. This may also explain why they have been shown to exhibit hormonal conditioning processes in a larger extent compared to other sports.

The particularities above-mentioned provide substantiation to the that FFT/HIFT may promote distinct responses when compared to other sports, and deserves specific studies.

Specific comments:

Line 33-34 – Better if reworded to “whether perturbations in immune parameters occur, even after one single bout, remains unclear”

Lines 43– I recommend using “‘a single Cindy’ workout session” instead of “‘Cindy’ workout.”, since the first expression better describes the experimentation in the present manuscript.

Lines 63-65 – Authors could also mention that HIFT regimens require both endurance and strength/resistance abilities, as well as an inherent irregularity as another hallmark characteristic of HIFT.

Line 91 – “ a single Cindy’ workout training session would promote perturbations in immune parameters” instead of “ ‘Cindy’ workout would promote perturbations in immune parameters, after a training session”.

Materials and methods

Overall comment:

I missed information regarding nutritional status. We know that pre-workout macronutrient intake (besides caffeine, creatine, and others) as well as chronic, daily caloric intake and macronutrient proportion may drive responses, even the acute ones. In case authors have not assessed this information, please openly describe in the discussion and or limitations of the study that these factors could also influence the responses, but were not obtained for the present study. In case authors did collect this data, please describe it.

Specific comments:

Lines 118-119 – Were experienced participants training for competitions? This would be interesting to be mentioned, since they change their level of effort when they are training for a competition, as part of the inherent but healthy intra- and inter-individual competitiveness of FFT/HIFT.

Line 123-125 – I find it very hard to find FFT/HIFT who do not take any type of “nutritional supplements composed of vitamins, minerals, or antioxidant

compounds” (item ‘d’ of exclusion criteria). It is important to remind that caffeine, for example, has sold evidence to enhance physical performance, and would therefore be a criteria of exclusion. However, earlier in the text authors recommend athletes to stay away from caffeine on the 48 hours before the beginning of the experiment. So we admit that some may were taking caffeine (in coffee or as caps) before, and were still included. Another examples are vitamin D and zinc, overly taken by almost all athletes (which I am particularly not against at all). Here, authors may described “supplements” more specifically (which supplements would cause exclusion?)

Lines 132-139 – The ability to perform all exercises is a premise to participate in the study, as far as I could understand. From this, I have two points:

1. Authors could make explicit the fact that participants should have been able to complete all physical executions, which makes lines 132-139 as a premise to be included; and

2. The description of any result is better suited in the first paragraphs of the results section, as well as the baseline characteristics, as shown in Table 1. I wo

Line 147a – Which was the interval between visits 3 and 4? This is important.

Line 147b – Please provide details on the morning before volunteers went to CrossFit gym. Were they recommended to have any specific sort of meal? More protein, more carbohydrate, or there were no specific recommendations? This is not such an issue in case athletes did not receive any specific nutritional recommendation, because they would intuitively follow their usual eating habits, as a “real life” study. But whether they received or not nutritional recommendations must be mentioned.

Lines 171-175 – Please describe which specific assay were employed for each parameter (which is not the information “All aforementioned variables were analysed using an automatic haematology analyser (Cell-Dyn; Abbott Laboratories, Abbott Park, IL, USA”).

Lines 182-183 – Even non-normal data was presented as mean and standard error? And why not both standard error and standard deviation (SD)?

Results

Overall comments:

1. Due to the extensive list of exclusion criteria (which I do agree with, except for a more specific list of supplements to exclude participation), prior to showing results, I would like to have a brief selection process (from 45 candidates, 4 were excluded because they were taking hormonal replacement, 3 because they were practicing irregularly…After the selection process, 23 participants completed the study.).

2. A sort of proportion between number of rounds and RPE could elicit a better picture of the differences between EXP and NOV.

3. I cannot read the exact number of biochemical parameters anywhere. They are not in the figures, tables, or text. Besides findings statistical significance and illustrating results in Figures (which gives a better understanding of the results), they must be described somewhere.

4. Neutrophil-to-lymphocyte ratio has emerged as a prognostic marker, although with distinct meanings between healthy and disease states. This is easy to calculate and would provide additional information.

5. Besides catecholamines, cortisol has been repeatedly reported to increase neutrophil mobilization, not only inhibiting lymphocytes overall activity. Then, at least part of acute neutrophil release could be secondary to cortisol increase. A “cortisol adjusted” neutrophil counting (lke neutrophil:cortisol ratio) can offer a view of cortisol-independent increase of neutrophilsm, which could be particularly useful for the 30-minute after exercise collect, since cortisol takes a little bit longer than catecholamines to induce increase in neutrophils.

6. In addition to the number of rounds, the mean total duration of the Cindy workout session should be reported, since some parameters are more strictly related to duration than intensity of exercise, and number of rounds are not necessarily linearly correlated with duration, since it also depends on the speed and duration of each round.

7. Adjustments of results for differences in body fat may provide additional information, since adipose tissue also releases IL-6 and may direct- and indirectly influence responses, while groups had significant differences between body fat. The same can be applied for the amount of muscle mass.

8. A sub-group analysis of sex-specific findings would be interesting to demonstrate trends in differences. One must consider that differences in male and female physiology encompass all organs, tissues, and metabolism. Sex-specific responses, particularly in terms of timing of response, could add useful information.

Discussion:

1. CK levels are more related to changes in training patterns (any change induces more prominent increase), rather than intensity. High-intensity regimens in those that were already used to these regimens tend to have lower increase of CK, compared to those who are experiencing high-intensity trainings for the first times. Interestingly, the fact that beginners had similar CK increase compared to experienced ones eludes to the fact that the ability to get more easily conditioned may occur early in FFT/HIFT, since beginners did not had enhanced CK increased compared to experienced ones.

2. Authors expand discussions to IL-6, dedicating a whole paragraph to this, without connecting IL-6 with the present findings of the study. Discussion should focus on the findings of the present study, and although they may mention on parameters not evaluated herein, at least they need to make a connection (what IL-6 increase, which likely happened in the athletes of the present study, has to do with the findings?)

3. Authors should consider extrapolate and make descriptive comparisons with sports of other modalities, including endurance, pure strength, and explosive (ball games, for example) sports. Are these exercise-specific responses, or responses seen in general? Is it there any peculiarity in the responses not found in other sports?

4. The sequence of the discussion is a but confusing. Authors come and go to markers (for example: lactate). Please reorder to provide a logical sequence.

Specific comments:

Lines 279-282 – Since changes in IL-6 and IL-10 have been described in the introduction, I would change the sentence for: “Although cytokines have not been analysed in the present study, it was previously demonstrated that FFT, such as a CrossFit® session, immune perturbations possibly due to changes in IL-6 and IL-10 blood levels occurred because exercise protocols caused high cardiovascular, metabolic, and hormonal demands, as observed in previous 282 studies [2,9,23,27,28]”

Lines 282-284 – This has been reported in the introduction. Also, describing results “4.1 and 14.4 pg/mL” should not be alone, but compared to pre-workout levels, and should include reference ranges. Please amend.

Lines 287-288 – In order to predict that similar responses in inflammatory markers occurred in the present protocol, similar responses between parameters measured in the present study and in the study described should be present, or a description of why similar responses would be expected, something like:

“because our protocol has similar characteristics of those studies that showed increase of IL-6 and IL-10 in response to exercise, we believe that similar responses…”

Lines 289-296 – Authors should comment on the still not fully elucidated differences between the muscle-released IL-6 (as a myokine) and adipose tissue-released IL-6 (as an adipokine), in terms of biological actions. Before that, it is essential to mention that the increase of IL-6 observed in previous studies is likely originated in the muscle tissue.

Also, the promotion of leucocyte adherence caused by IL-6 would lead to decrease in WBC blood levels, since these would be attached to endothelium. Please explore this point.

Lines 197-307 – While catecholamines actions prevail over cortisol during and right after exercise, 30 minutes after the end of the training session cortisol actions prevails, while catecholamines faster return to normal levels. Cortisol promotes a pro-neutrophil anti-lymphocyte environment, leading to increased neutrophil:lymphocyte ratio (and this is why I mentioned the importance of additing this ratio as a parameter). This well explains the findings described in lines 197-198, at a larger extent than the explanation on lines 300-302.

Lines 313-314 – There is a counterargument to the point in lines 313-314, that should be mentioned: because of the higher number of rounds, the duration of the training session of EXP athletes was likely longer than NOV. Cortisol tends to be released in a training duration-manner, which means that EXP were more exposed to cortisol compared to NOV, particularly if we calculate the area under the curve (AUC) of its release (as mentioned by authors later, in lines 371-372). Hence, one would expect that lymphocyte count would be lower, not higher, in the EXP group, due to cortisol inhibitory effects.

This should be explored in the discussion.

Line 336 – How were participants exposed to an exact 20-minute duration intervention, if the number of rounds differed between them? (at least from what I understood, CK was collected in visits 4 and 5 – and in visit 4, which was the training session, I have not seen anything specifying the training duration. Otherwise number of sessions would be compromised and limited.) Please justify.

Lines 342-343 – Release of CK and myoglobin in the circulation due to damaged muscle tissue is an physiological adaptation to exercise. Authors should specify in which point and extent (if any) this muscle breakdown becomes pathological (rhabdomuolysis). A suggestion:

“Rhabdomyolysis is a condition in which the excessive amount of damaged muscle tissue breaks down its intracellular contents, and abnormal CK and myoglobin levels in relation to those expected for responses to exercise are released into the circulation, leading to secondary clinical and biochemical complications”.

Lines 350-354 – The level of increase of CK levels does not depend on the amount of work alone, but on the amount of work performed in comparison to the previous amount of work performed in previous trainings. EXP athletes performed more work because they were used to perform more work before. Hence, the proportion between work performed in the present analysis and usually performed before were possibly similar between EXP and NOV. This is likely the most suitable explanation for the lack of differences in the amplitude of CK increase.

Lines 387-388 – That would have been interesting if authors had collected lactate 24 hours after training. This would better demonstrate potential differences in lactate clearance speed between groups, as a marker of recovery speed.

Line 409 – “Regardless of experience and conditioning level”

Conclusion

Overall comment:

The way conclusion is written, it seems that there is no novelty in the present study, since muscle damage and stress markers are expected to occur in response to any minimally intense activity. A suggestion:

“This is the first report that a single FFT session elicited significant acute perturbations in WBC counting, stress markers, and muscle tissue, in an analogue manner than other high-intensity training regimens. Noteworthy, those more experienced exhibited greater lymphocyte and cortisol responses than novice ones.”

Specific comments:

Line 412 – Remove the expression “In conclusion,” Start directly with “One single..”

Lines 413-414 – “caused muscle damage”, not “increased”, because we presume that muscle damage was absent before.

Reviewer #2: This study aimed to examine acute leucocyte, muscle damage, and stress marker responses following a single bout of CrossFit and to compare the results between novice and experienced participants. To this author made a comparison between novice (3-8 months of experience; n = 10) and experienced (≥18 months; n = 13) subjects after ‘Cindy’ workout.

On the basis of their results, the authors concluded that the ‘Cindy’ workout elicited a significant perturbation in white blood cell (WBC) count, plasma creatine kinase activity, blood cortisol level, and lactate counts with increased muscle damage and stress markers and that experienced participants showed greater responses than novice.

The article is interesting and well-structured. Nevertheless, the quality of the paper needs to be improved and some corrections and implementations should be provided.

-The paper is too long, particularly the discussion section, and must be shortened.

-What is the reason for classified the subjects in EXP only through the months of training? For example, if an individual had 2 years of experience but cannot perform on single repetition of pull-up? Please explain.

-Can the authors perform a correlation between changes in immune, hormonal, and metabolic response with volume of repetitions? This can explain the higher hormonal and immune response in EXP as compared to NOV.

-Can the authors explain why the VO2 of experienced volunteers is below those reported in previous studies? Bellar et al., Biol Sport. 2015. Tibana et al., Sports. 2019; Sousa et al., 2016 - Journal of exercise physiology online; Butcher et al., 2015 Open access journal of sports medicine

-Can the authors perform a correlation between changes in immune, hormonal, and metabolic response with physical fitness tests (cardiovascular and muscle strength)? Are the subjects with better physical conditioning less responsive to muscle perturbation?

It is important to note that a functional fitness training session does not only include metabolic conditioning (for example Cindy), but includes the development of strength, power and gymnastics.

-The author can include some recent articles to increase the quality of the discussion:

Posnakidis et al. High-Intensity Functional Training Improves Cardiorespiratory Fitness and Neuromuscular Performance Without Inflammation or Muscle Damage. 2020

Falk Neto et al. Session RPE is a superior method to monitor internal training loads of functional fitness training sessions performed at different intensities when compared to training impulse. 2020.

Mangine et al. Physiological Differences Between Advanced CrossFit Athletes, Recreational CrossFit Participants, and Physically-Active Adults. 2020.

Poderoso et al. Gender Differences in Chronic Hormonal and Immunological Responses to CrossFit. 2019

Tibana RA et al. Is Perceived Exertion a Useful Indicator of the Metabolic and Cardiovascular Responses to a Metabolic Conditioning Session of Functional Fitness? 2019.

Tibana RA et al. Lactate, Heart Rate and Rating of Perceived Exertion Responses to Shorter and Longer Duration CrossFit® Training Sessions

Reviewer #3: The manuscript is very well done. Additionally, there is little scientific literature addressing these types of stress markers, muscle damage, and white blood cells in Crossfit. For this reason, I think it has the potential to be published in one.

The introduction follows a common thread with the objectives statement.

The research design is appropriate and well described.

The results are well explained and easy to understand. And the discussion of the results addresses each of the variables analyzed.

Results

P. 10, line 228 There were no differences between immediately after, 30 min, and 24 h after exercise time points (p < 0.01). Modify by p > 0.01.

**********

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Reviewer #1: Yes: Flavio A. Cadegiani

Reviewer #2: Yes: Ramires A. Tibana

Reviewer #3: No

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13 Sep 2020

The responses for each reviewer comments are below as in the file "response to reviewers"

Reviewer #1:

General comments:

1. This is a simple yet well thought study. Authors found a gap on a very basic knowledge and explored it appropriately. These are those kind of basic studies that are more than necessary to better characterize the effects of this specific sport, with interesting peculiarities.

Answer: Dear reviewer, we are very grateful for all the notes and suggestions proposed, as they certainly contributed to the improvement of the quality of our study. We were very excited about your words.

2. “Functional Fitness” has also been largely used as “High Intensity Functional Training” HIFT). Since its main representative is the branded sport “CrossFit”, it was kind of challenging to uniformize a generic name. I recommend authors to change for “HIFT” throughout the manuscript.

Answer: We agreed and changed it according to the suggestion. Throughout the entire manuscript the term “Functional Fitness” was replaced by High-Intensity Functional Training” (HIFT).

3. Authors should be careful when using the brand name (CrossFit). Its previous owners of the brand (the brand CrossFit has been recently sold) were threatening several research groups that attempted to describe scientific reports of CrossFit, and copiously have attempted to retract studies on “CrossFit”.

Answer: Thanks for the alert. We reduced the use of the term “CrossFit”, being included only in sections where it was considered extremely necessary.

Introduction

Overall comment:

I suggest a brief explanation on why FFT/HIFT may elicit different effects than those of other sports, by comparing their characteristics, which the most fundamental ones are:

1. FFT/HIFT mixes endurance, explosion, and strength within the same sport, exercise, at the same time, unlike any other sport, and may be the only activity that requires all domains of physical conditioning.

2. There are not patterns or predictable sequence of exercises as well as the characteristics of exercise execution. This leads to higher caloric expenditure since It does not allow muscles to learn patterns and “predict” the following demand patterns. Hence, organism is constantly getting adapted. Perhaps, FFT/HIFT practitioners may develop a sort of an ability to “condition faster in conditioning processes”, i.e., their bodies develop specific conditioning abilities faster than any other sport, as they are constantly submitted to “unexpected” new physical demands. This may also explain why they have been shown to exhibit hormonal conditioning processes in a larger extent compared to other sports.

The particularities above-mentioned provide substantiation to the that FFT/HIFT may promote distinct responses when compared to other sports, and deserves specific studies.

Answer: We agreed and changed it, according to the suggestion. We added a reference (Feito Y, Heinrich K, Butcher S, Poston W. High-Intensity Functional Training (HIFT): Definition and Research Implications for Improved Fitness. Sports. 2018;6: 76. doi:10.3390/sports6030076.) that discusses the definition of HIFT to better clarify the concepts mentioned by the reviewer. Lines 65-69

Specific comments:

Lines 33-34 – Better if reworded to “whether perturbations in immune parameters occur, even after one single bout, remains unclear”.

Answer: We agreed and changed the sentence. Lines 33-34

Line 43– I recommend using “‘a single Cindy’ workout session” instead of “‘Cindy’ workout.”, since the first expression better describes the experimentation in the present manuscript.

Answer: We agreed and changed the words throughout the text. First replacement in Line 43.

Lines 63-65 – Authors could also mention that HIFT regimens require both endurance and strength/resistance abilities, as well as an inherent irregularity as another hallmark characteristic of HIFT.

Answer: We agreed and added a sentence considering the observation in the paragraph, referencing the due study (Cadegiani FA, Kater CE, Gazola M. Clinical and biochemical characteristics of high-intensity functional training (HIFT) and overtraining syndrome: findings from the EROS study (The EROS-HIFT). J Sports Sci. 2019;37: 1296–1307. doi:10.1080/02640414.2018.1555912.) Lines 70-72

Line 91 – “a single Cindy’ workout training session would promote perturbations in immune parameters” instead of “ ‘Cindy’ workout would promote perturbations in immune parameters, after a training session”.

Answer: We agreed and changed the words in the text. Lines 99-100

Materials and methods

Overall comment:

I missed information regarding nutritional status. We know that pre-workout macronutrient intake (besides caffeine, creatine, and others) as well as chronic, daily caloric intake and macronutrient proportion may drive responses, even the acute ones. In case authors have not assessed this information, please openly describe in the discussion and or limitations of the study that these factors could also influence the responses, but were not obtained for the present study. In case authors did collect this data, please describe it.

Answer: We agree that pre-workout macronutrient intake as well as chronic daily caloric intake and macronutrient proportion may drive responses. So, we have additional information regarding nutritional status that have been included in the revised manuscript, and we believe they can enrich the work.

Pre-workout meal: Participants were instructed on the importance of pre-workout diet, and for that reason, a standardized meal was provided for that moment. Even two participants were excluded from the study because they did not eat the standardized meal. In the manuscript originally sent, we had mentioned the characteristics of the pre-workout intake (lines 164-167 / Lines 183-186 in the revised manuscript): “One hour before the training session, the volunteers consumed a standardized breakfast (approximately 320–350 calories) with a protein to fat to carbohydrate ratio of 20:35:45 (protein, fat, carbohydrate as percentage)”. In the revised version of the manuscript, we include the following complementation: these percentages culminated in the intake of approximately 40g of carbohydrates, 17.5g of protein and 13g of fat (lines 186-187). We hope that this information is satisfactory.

Chronic energy and macronutrient intake: our initial proposal also included this type of analysis, and for that, participants were asked to record three non-consecutive days of their food intake, for further evaluation of energy intake and macronutrients using the software “Dietpro” version 5.1; however, adherence to the delivery of such forms was only 52.2%, that is, of the 23 participants, only 12 delivered the completed forms, being 9 from the group EXP and only 3 from NOV. For this reason, we had given up on including such data in the publication, however, at the suggestion of the reviewers, we included the data, and pointed out its limitations, with regard to the reduced number of participants in the beginning group. Nutritional data analysis (12 participants) was inserted in the results section (table 4).

Due to the inclusion of the information regarding nutritional status, changes were made to the manuscript, in the following sections:

Materials and methods: Lines 117-122

Results: Table 4 has been included, as well as its description in lines 348-353.

Discussion: Lines 520-536

Limitations: Lines 542-544

Lines 118-119 – Were experienced participants training for competitions? This would be interesting to be mentioned, since they change their level of effort when they are training for a competition, as part of the inherent but healthy intra- and inter-individual competitiveness of FFT/HIFT.

Answer: The experienced participants were not training for competitions. We added the sentence in the paragraph showing the profile of experienced participants. Lines 155-157

Line 123-125 – I find it very hard to find FFT/HIFT who do not take any type of “nutritional supplements composed of vitamins, minerals, or antioxidant compounds” (item ‘d’ of exclusion criteria). It is important to remind that caffeine, for example, has sold evidence to enhance physical performance, and would therefore be a criteria of exclusion. However, earlier in the text authors recommend athletes to stay away from caffeine on the 48 hours before the beginning of the experiment. So we admit that some may were taking caffeine (in coffee or as caps) before, and were still included. Another examples are vitamin D and zinc, overly taken by almost all athletes (which I am particularly not against at all). Here, authors may described “supplements” more specifically (which supplements would cause exclusion?).

Answer: We appreciate your observation. We agree that there was a lack of details in the material and methods section to clarify these informations. It was really difficult to find participants who were not consuming supplements that contained: a) antioxidant compounds in last six weeks and b) ergogenic compounds in last four months; for this reason, it is important to mention that this was the criterion that most excluded volunteers from the research (82 exclusions). The description of the selection process was included at the beginning of the results section (lines 226-227).

Supplements considered as exclusion criteria were those containing antioxidant compounds in last six weeks, as well as those considered ergogenic (ISSN, 2018), such as caffeine, creatine, beta-alanine, nitrate and bicarbonates. The consumption of any of these ergogenic supplements in the last four months would exclude the volunteer. This information was included in the revised version of the manuscript on lines 142-145.

About caffeine, specifically, cited in line 112 of the original manuscript: participants were advised to avoid caffeine, but we were referring to caffeine-rich drinks or food (e.g., tea, coffee, and chocolate), as they are products capable of reducing body hydration. This information has been included in the Line 122.

The term “supplement” for caffeine was not used in line 112 of the original manuscript, as this consumption was considered an exclusion criterion, the authors assumed that there would be no participant with this profile.

Lines 132-139 – The ability to perform all exercises is a premise to participate in the study, as far as I could understand. From this, I have two points:

1. Authors could make explicit the fact that participants should have been able to complete all physical executions, which makes lines 132-139 as a premise to be included; and

Answer: We agreed and changed it according to the suggestion. We added as a premise for participation in the study the inclusion criteria. Line 136.

2. The description of any result is better suited in the first paragraphs of the results section, as well as the baseline characteristics, as shown in Table 1.

Answer: We agreed and changed it according to the suggestion. We moved the description of the baseline characteristics and “Table 1” to the results section. Lines 235-239.

Line 147a – Which was the interval between visits 3 and 4? This is important.

Answer: We agreed and added this information. Line 165

Line 147b – Please provide details on the morning before volunteers went to CrossFit gym. Were they recommended to have any specific sort of meal? More protein, more carbohydrate, or there were no specific recommendations? This is not such an issue in case athletes did not receive any specific nutritional recommendation, because they would intuitively follow their usual eating habits, as a “real life” study. But whether they received or not nutritional recommendations must be mentioned.

Answer: We agree that pre-workout macronutrient intake may drive responses. Participants were instructed on the importance of pre-workout diet, and for that reason, a standardized meal was provided for that moment. In the original manuscript (lines 164-167), we had mentioned the characteristics of the pre-workout intake: “One hour before the training session, the volunteers consumed a standardized breakfast (approximately 320–350 calories) with a protein to fat to carbohydrate ratio of 20:35:45 (protein, fat, carbohydrate as percentage)” (Lines 183-186 in the revised manuscript). In the revised version of the manuscript, we include the following complementation: these percentages culminated in the intake of approximately 40g of carbohydrates, 17.5g of protein and 13g of fat (lines 186-187). We hope that this information is satisfactory. Lines 186-187 in the revised manuscript.

Lines 171-175 – Please describe which specific assay were employed for each parameter (which is not the information “All aforementioned variables were analysed using an automatic haematology analyser (Cell-Dyn; Abbott Laboratories, Abbott Park, IL, USA”).

Answer: We agreed and added this information, describing in detail the specific assay for these analyses. Lines 195-201.

Lines 182-183 – Even non-normal data was presented as mean and standard error? And why not both standard error and standard deviation (SD)?

Answer: Non-normal data were presented as mean to allow comparison with other studies (Mangine et al., 2020; Poderoso et al., 2019; and Tibana et al., 2019). As suggested, we have included standard deviation in all the tables. Additionally, we include a new table (table 5) in “supporting information” with SD data referring to the figures.

Results

Overall comments:

1. Due to the extensive list of exclusion criteria (which I do agree with, except for a more specific list of supplements to exclude participation), prior to showing results, I would like to have a brief selection process (from 45 candidates, 4 were excluded because they were taking hormonal replacement, 3 because they were practicing irregularly…After the selection process, 23 participants completed the study).

Answer: We agreed and added this information, describing in detail the selection process. Lines 224-230.

2. A sort of proportion between number of rounds and RPE could elicit a better picture of the differences between EXP and NOV.

Answer: We agreed and added this information in table 2 (Line 250). The inclusion of this result led to the addition of a sentence in the discussion section (Lines 388-389).

3. I cannot read the exact number of biochemical parameters anywhere. They are not in the figures, tables, or text. Besides findings statistical significance and illustrating results in Figures (which gives a better understanding of the results), they must be described somewhere.

Answer: We agreed and inserted a new table (table 5). Table 5 is available at supporting information

4. Neutrophil-to-lymphocyte ratio has emerged as a prognostic marker, although with distinct meanings between healthy and disease states. This is easy to calculate and would provide additional information.

Answer: We agreed and added this information. It was calculated the neutrophil/lymphocyte ratio in both groups, and these results were described in table 3 and cited in lines 284-288. The inclusion of these results led to the addition of a paragraph in the discussion section (lines 450-454)

5. Besides catecholamines, cortisol has been repeatedly reported to increase neutrophil mobilization, not only inhibiting lymphocytes overall activity. Then, at least part of acute neutrophil release could be secondary to cortisol increase. A “cortisol adjusted” neutrophil counting (lke neutrophil:cortisol ratio) can offer a view of cortisol-independent increase of neutrophilsm, which could be particularly useful for the 30-minute after exercise collect, since cortisol takes a little bit longer than catecholamines to induce increase in neutrophils.

and and neutrophils-to-cortisol ratio

Answer: We agreed and added this information. It was calculated the neutrophil/cortisol ratio in both groups, and these results were described in table 3 and cited in lines 288-290. The inclusion of these results led to the addition of a paragraph in the discussion section (line 454-458)

6. In addition to the number of rounds, the mean total duration of the Cindy workout session should be reported, since some parameters are more strictly related to duration than intensity of exercise, and number of rounds are not necessarily linearly correlated with duration, since it also depends on the speed and duration of each round.

Answer: We agreed with the observation of the esteemed reviewer. In this case, we put as a limitation of the study not having exactly controlled the real effort time of each participant (line 541)

7. Adjustments of results for differences in body fat may provide additional information, since adipose tissue also releases IL-6 and may direct- and indirectly influence responses, while groups had significant differences between body fat. The same can be applied for the amount of muscle mass.

Answer: We agreed with the observation. The Spearman product moment correlation was used, but no correlation was found between body composition and cortisol and WBC. If we had analyzed IL-6, we might have obtained a better correlation.

8. A sub-group analysis of sex-specific findings would be interesting to demonstrate trends in differences. One must consider that differences in male and female physiology encompass all organs, tissues, and metabolism. Sex-specific responses, particularly in terms of timing of response, could add useful information.

Answer: We agreed with the observation. We will soon be submitting a new manuscript comparing men and women.

Discussion:

1. CK levels are more related to changes in training patterns (any change induces more prominent increase), rather than intensity. High-intensity regimens in those that were already used to these regimens tend to have lower increase of CK, compared to those who are experiencing high-intensity trainings for the first times. Interestingly, the fact that beginners had similar CK increase compared to experienced ones eludes to the fact that the ability to get more easily conditioned may occur early in FFT/HIFT, since beginners did not had enhanced CK increased compared to experienced ones.

Answer: We agreed and appreciate so much the collaboration. We explored this point of view. (Lines 493-498)

2. Authors expand discussions to IL-6, dedicating a whole paragraph to this, without connecting IL-6 with the present findings of the study. Discussion should focus on the findings of the present study, and although they may mention on parameters not evaluated herein, at least they need to make a connection (what IL-6 increase, which likely happened in the athletes of the present study, has to do with the findings?)

Answer: We agreed with the observation. We reformulated the paragraphs referring to the discussion about IL-6, establishing a greater connection with our findings (Lines 428-444)

3. Authors should consider extrapolate and make descriptive comparisons with sports of other modalities, including endurance, pure strength, and explosive (ball games, for example) sports. Are these exercise-specific responses, or responses seen in general? Is it there any peculiarity in the responses not found in other sports?

Answer: Excellent observation. Our opinion is that it would not be appropriate to extrapolate to other sports or exercises because the protocol used in the present study is quite different. From a biomechanical point of view, no sport (individual or collective) resembles Cindy. From the metabolic and cardiovascular point of view, it is possible that some endurance sports are close to what was observed in the present study, but with different duration. In this sense, we chose not to extrapolate, agreeing with Cadegiani et al. 2019(J Sports Sci), who highlighted the characteristics of HIFT as follows: "Compared to other sport modalities, CF has unique patterns, as it requires multiple abilities, given the complexity and irregularity of its activities”.

4. The sequence of the discussion is a but confusing. Authors come and go to markers (for example: lactate). Please reorder to provide a logical sequence.

Answer: We agreed and reordered the paragraphs, improving the fluidity of the reading.

Specific comments:

Lines 279-282 – Since changes in IL-6 and IL-10 have been described in the introduction, I would change the sentence for: “Although cytokines have not been analysed in the present study, it was previously demonstrated that FFT, such as a CrossFit® session, immune perturbations possibly due to changes in IL-6 and IL-10 blood levels occurred because exercise protocols caused high cardiovascular, metabolic, and hormonal demands, as observed in previous studies [2,9,23,27,28]”

Answer: In order to focus on discussing the study's findings, we chose to reduce the paragraphs related to IL-6 and IL-10. Thus, this paragraph above was excluded from the manuscript (Lines 428-444), since the relationship between IL-6 and our findings was described in lines 428-444.

Lines 282-284 – This has been reported in the introduction. Also, describing results “4.1 and 14.4 pg/mL” should not be alone, but compared to pre-workout levels, and should include reference ranges. Please amend.

Answer: As we did not analyze interleukins in our study, as suggested by the esteemed reviewer, we decided not to detail the concentrations of this parameter in our discussion, as we would not be able to compare them. So, the paragraph was excluded from the manuscript.

Lines 287-288 – In order to predict that similar responses in inflammatory markers occurred in the present protocol, similar responses between parameters measured in the present study and in the study described should be present, or a description of why similar responses would be expected, something like:

“because our protocol has similar characteristics of those studies that showed increase of IL-6 and IL-10 in response to exercise, we believe that similar responses…”

Answer: In order to focus on discussing the study's findings, we chose to reduce the paragraphs related to IL-6 and IL-10. Thus, this paragraph was excluded from the manuscript.

Lines 289-296 – Authors should comment on the still not fully elucidated differences between the muscle-released IL-6 (as a myokine) and adipose tissue-released IL-6 (as an adipokine), in terms of biological actions. Before that, it is essential to mention that the increase of IL-6 observed in previous studies is likely originated in the muscle tissue.

Also, the promotion of leucocyte adherence caused by IL-6 would lead to decrease in WBC blood levels, since these would be attached to endothelium. Please explore this point.

Answer: Some details on IL-6 synthesis were discussed in lines 428-444.

Lines 297-307 – While catecholamines actions prevail over cortisol during and right after exercise, 30 minutes after the end of the training session cortisol actions prevails, while catecholamines faster return to normal levels. Cortisol promotes a pro-neutrophil anti-lymphocyte environment, leading to increased neutrophil:lymphocyte ratio (and this is why I mentioned the importance of additing this ratio as a parameter). This well explains the findings described in lines 197-198, at a larger extent than the explanation on lines 300-302.

Answer: We appreciate the great suggestion, and have included this important parameter in our discussion. We believe that it has added relevant value to our study (Lines 447-454).

Lines 313-314 – There is a counterargument to the point in lines 313-314, that should be mentioned: because of the higher number of rounds, the duration of the training session of EXP athletes was likely longer than NOV. Cortisol tends to be released in a training duration-manner, which means that EXP were more exposed to cortisol compared to NOV, particularly if we calculate the area under the curve (AUC) of its release (as mentioned by authors later, in lines 371-372). Hence, one would expect that lymphocyte count would be lower, not higher, in the EXP group, due to cortisol inhibitory effects.

This should be explored in the discussion.

Answer: We appreciate the great suggestion, and have included this important parameter in our results (figure 4 and Lines 332-333) and discussion (Line 408). We believe that it has added relevant value to our study.

Line 336 – How were participants exposed to an exact 20-minute duration intervention, if the number of rounds differed between them? (at least from what I understood, CK was collected in visits 4 and 5 – and in visit 4, which was the training session, I have not seen anything specifying the training duration. Otherwise number of sessions would be compromised and limited.) Please justify.

Answer: The Cindy workout is based on Crossfit's AMRAP (As Many Rounds As Possible) concept. In this type of training, the practitioner has a predetermined duration to perform the greatest number of rounds / repetitions of a given sequence of exercises. Therefore, as much as they had a different number of rounds, the total duration of the effort was 20 minutes. We were very strict in relation to the collection control, timing exactly for each participant the exact time during the intervention. To date, we have found no studies that have monitored the real time of effort during these 20 minutes of Cindy workout. This observation we include in the limitations of the present study (Line 541).

Lines 342-343 – Release of CK and myoglobin in the circulation due to damaged muscle tissue is an physiological adaptation to exercise. Authors should specify in which point and extent (if any) this muscle breakdown becomes pathological (rhabdomuolysis). A suggestion:

“Rhabdomyolysis is a condition in which the excessive amount of damaged muscle tissue breaks down its intracellular contents, and abnormal CK and myoglobin levels in relation to those expected for responses to exercise are released into the circulation, leading to secondary clinical and biochemical complications”.

Answer: Suggestion accepted. The paragraph has been included (Lines 503-507)

Lines 350-354 – The level of increase of CK levels does not depend on the amount of work alone, but on the amount of work performed in comparison to the previous amount of work performed in previous trainings. EXP athletes performed more work because they were used to perform more work before. Hence, the proportion between work performed in the present analysis and usually performed before were possibly similar between EXP and NOV. This is likely the most suitable explanation for the lack of differences in the amplitude of CK increase.

Answer: Suggestion accepted. The paragraph has been included (Lines 511-517)

Lines 387-388 – That would have been interesting if authors had collected lactate 24 hours after training. This would better demonstrate potential differences in lactate clearance speed between groups, as a marker of recovery speed.

Answer: This is a very interesting suggestion for future studies. We inserted it as a limitation of the study in lines 539-541

Line 409 – “Regardless of experience and conditioning level”

Answer: Suggestion accepted. The sentence has been included. (Lines 555-556)

Conclusion

Overall comment:

The way conclusion is written, it seems that there is no novelty in the present study, since muscle damage and stress markers are expected to occur in response to any minimally intense activity. A suggestion:

“This is the first report that a single FFT session elicited significant acute perturbations in WBC counting, stress markers, and muscle tissue, in an analogue manner than other high-intensity training regimens. Noteworthy, those more experienced exhibited greater lymphocyte and cortisol responses than novice ones.”

Answer: We agreed and replace the paragraph. We appreciate so much for the suggestion (Lines 560-563)

Specific comments:

Line 412 – Remove the expression “In conclusion,” Start directly with “One single..” (Line 560)

Answer: We agreed and made the modification.

Lines 413-414 – “caused muscle damage”, not “increased”, because we presume that muscle damage was absent before.

Answer: We agreed and made the modification.

Reviewer #2: This study aimed to examine acute leucocyte, muscle damage, and stress marker responses following a single bout of CrossFit and to compare the results between novice and experienced participants. To this author made a comparison between novice (3-8 months of experience; n = 10) and experienced (≥18 months; n = 13) subjects after ‘Cindy’ workout.

On the basis of their results, the authors concluded that the ‘Cindy’ workout elicited a significant perturbation in white blood cell (WBC) count, plasma creatine kinase activity, blood cortisol level, and lactate counts with increased muscle damage and stress markers and that experienced participants showed greater responses than novice.

The article is interesting and well-structured. Nevertheless, the quality of the paper needs to be improved and some corrections and implementations should be provided.

Answer: Dear reviewer, we appreciate all the suggestions. We are sure that the quality of the paper has been greatly enriched. We would like to highlight that a new revision of the English language was made in the entire manuscript.

- The paper is too long, particularly the discussion section, and must be shortened.

Answer: We agreed and reorganize the fluidity of the paragraphs in the discussion section. Certainly, the discussion became more objective, however due to the inclusion of new analyzes requested by the reviewers (RPE-Number of rounds ratio; NLR = Neutrophil / Lymphocyte ratio; NCR: Neutrophil / Cortisol ratio; area under the curve (AUC) of cortisol; Dietary analysis; and correlations) the discussion became more extensive. But we believe that it has added relevant value to our study.

-What is the reason for classified the subjects in EXP only through the months of training? For example, if an individual had 2 years of experience but cannot perform on single repetition of pull-up? Please explain.

Answer: The question is very pertinent. For the classification of experienced and novice participants, we follow the criteria listed below:

i) We follow the same criteria as the recent publication by Butcher et al. Relative Intensity Of Two Types Of Crossfit Exercise: Acute Circuit And High-Intensity Interval Exercise. J Fit Res. 2015., which adopted these intervals time.

ii) As for performing a single pull up, in the experienced group, all 13 performed without modifications, that is, they performed both pull up and push up without modifications. In the NOV group, of the 10 participants, 7 required modification in the pull-up or push up, or in both.

-Can the authors perform a correlation between changes in immune, hormonal, and metabolic response with volume of repetitions? This can explain the higher hormonal and immune response in EXP as compared to NOV.

Answer: We correlated the number of rounds with the hormonal parameters and the immune system. We found only the significant correlations between:

- Rounds and leukocytes in post-ex (r = 0.43; p = 0.040)

- Rounds and lymphocytes in post-ex (r = 0.55; p = 0.006)

- Rounds and cortisol in post-ex (r = 0.53; p = 0.010)

These results have been inserted in the paragraphs of the results section in lines 310-311 and 333-334. The inclusion of these results led to the addition of a sentence in the discussion section (lines 410-412 and 426-427).

-Can the authors explain why the VO2 of experienced volunteers is below those reported in previous studies? Bellar et al., Biol Sport. 2015. Tibana et al., Sports. 2019; Sousa et al., 2016, Journal of exercise physiology online; Butcher et al., 2015 Open access journal of sports medicine.

Answer: The reasons for having found VO2max values below the aforementioned studies are:

i) Characteristics of the participants in our study. Unlike the other studies, the participants in the present study were not engaged in competitions. Even the most experienced were recreational CrossFit practitioners. Although the city of Aracaju is the capital of the State of Sergipe, recreational, amateur and competitive sports receive little incentive, which makes it difficult to find highly trained or competitive people. We highlight this characteristic of our sample in the lines. Lines 155-157 and 367-368.

ii) We use the yo-yo test to predict VO2max, that is, an indirect test. According to previous publications (Stojanovic et al., 2016, Montenegrin Journal of Sports Science and Medicine; Martínez-Lagunas and Hartmann, 2014, International Journal of Sports Physiology and Performance), the yo-yo test generally underestimate VO2max. The aforementioned studies used a gas analyzer. We highlight this observation in the discussion section, as study limitations. Lines 541-542.

-Can the authors perform a correlation between changes in immune, hormonal, and metabolic response with physical fitness tests (cardiovascular and muscle strength)? Are the subjects with better physical conditioning less responsive to muscle perturbation?

Answer: We correlated physical fitness tests (cardiovascular and muscle strength) with hormonal parameters, immune systems and stress markers. We found no significant correlations in any of the combinations.

- It is important to note that a functional fitness training session does not only include metabolic conditioning (for example Cindy), but includes the development of strength, power and gymnastics.

Answer: We agreed and added the excerpt highlighting that the HIFT has, in addition to metabolic conditioning, exercises aimed at strength, power and gymnastics, as a study limitation (Lines 544-547).

-The author can include some recent articles to increase the quality of the discussion:

Answer: Dear reviewer, we appreciate so much your collaboration. We have included the excellent references suggested below.

- Posnakidis et al. High-Intensity Functional Training Improves Cardiorespiratory Fitness and Neuromuscular Performance Without Inflammation or Muscle Damage. 2020

- Falk Neto et al. Session RPE is a superior method to monitor internal training loads of functional fitness training sessions performed at different intensities when compared to training impulse. 2020.

- Mangine et al. Physiological Differences Between Advanced CrossFit Athletes, Recreational CrossFit Participants, and Physically-Active Adults. 2020.

- Poderoso et al. Gender Differences in Chronic Hormonal and Immunological Responses to CrossFit. 2019

- Tibana RA et al. Is Perceived Exertion a Useful Indicator of the Metabolic and Cardiovascular Responses to a Metabolic Conditioning Session of Functional Fitness? 2019.

- Tibana RA et al. Lactate, Heart Rate and Rating of Perceived Exertion Responses to Shorter and Longer Duration CrossFit® Training Sessions

Reviewer #3: The manuscript is very well done. Additionally, there is little scientific literature addressing these types of stress markers, muscle damage, and white blood cells in Crossfit. For this reason, I think it has the potential to be published in one.

The introduction follows a common thread with the objectives statement.

The research design is appropriate and well described.

The results are well explained and easy to understand. And the discussion of the results addresses each of the variables analyzed.

Answer: Dear reviewer, we are very grateful for the compliments to our manuscript.

Results

P. 10, line 228 There were no differences between immediately after, 30 min, and 24 h after exercise time points (p < 0.01). Modify by p > 0.01.

Answer: Thank you. We change accordingly (Line 316)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 Nov 2020

PONE-D-20-16542R1

Acute leucocyte, muscle damage, and stress marker responses to high-intensity functional training

PLOS ONE

Dear Dr. Gomes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the point raised during the review process by reviewer 2.

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We look forward to receiving your revised manuscript.

Kind regards,

Pedro Tauler, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have fully addressed my suggestions, and raised the quality of their manuscript. Very few studies on HIFT in the world have the quality of the present paper. Congratulations!

Reviewer #2: Congratulate the authors for the effort done in improving the paper and addressing my concerns. Nevertheless, I still think some issues need clarity.

"We believe that the difference in VO2max is what mainly caused the EXP group to perform more rounds and maintain the same alterations in lactate concentration, as the maximum strength level revealed no differences between groups. Although the NOV group had only 3 to 8 months of experience with HIFT, they were all well versed in strength training."

This paragraph is not supported by scientific evidence. Butcher et al., (2015) concluded that CrossFit benchmark Cindy performance cannot be predicted by VO2. Perhaps the difference between EXP and NOV is explained by Local Muscle Endurance.

Butcher et al. Open Access J Sports Med. 2015 Jul 31;6:241-7. d

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Flavio A. Cadegiani, MD, MSc, Ph.D

Reviewer #2: Yes: Ramires Alsamir Tibana

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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16 Nov 2020

PONE-D-20-16542R1

Acute leucocyte, muscle damage, and stress marker responses to high-intensity functional training

Reviewer #1:

General comments:

Authors have fully addressed my suggestions, and raised the quality of their manuscript. Very few studies on HIFT in the world have the quality of the present paper. Congratulations!

Answer: Dear reviewer, we are flattered by your words. It is extremely valuable for us to receive your congratulations. Your contributions were essential for us to reach this level of quality.

Reviewer #2:

General comments:

Congratulate the authors for the effort done in improving the paper and addressing my concerns. Nevertheless, I still think some issues need clarity.

Answer: Dear reviewer, we are very grateful for the considerations. It is a great honor for us to receive your compliments.

Discussion

P. 16, Line paragraph - 390 "We believe that the difference in VO2max is what mainly caused the EXP group to perform more rounds and maintain the same alterations in lactate concentration, as the maximum strength level revealed no differences between groups. Although the NOV group had only 3 to 8 months of experience with HIFT, they were all well versed in strength training."

This paragraph is not supported by scientific evidence. Butcher et al., (2015) concluded that CrossFit benchmark Cindy performance cannot be predicted by VO2. Perhaps the difference between EXP and NOV is explained by Local Muscle Endurance.

Butcher et al. Open Access J Sports Med. 2015 Jul 31;6:241-7. D

Answer: We agree with the observation and thank you for the indication of the article "Do physiological measures predict selected CrossFit (®) benchmark performance?”. Therefore, we reformulated the lines mentioned according to the information described in that article.

(Line 392-406)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

19 Nov 2020

Acute leucocyte, muscle damage, and stress marker responses to high-intensity functional training

PONE-D-20-16542R2

Dear Dr. João Henrique Gomes,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Pedro Tauler, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Ramires A. Tibana

23 Nov 2020

PONE-D-20-16542R2

Acute leucocyte, muscle damage, and stress marker responses to high-intensity functional training

Dear Dr. Gomes:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Pedro Tauler

Academic Editor

PLOS ONE