Effect of caffeine on neuromuscular function following eccentric-based exercise.

This study investigated the effect of caffeine on neuromuscular function, power and sprint performance during the days following an eccentric-based exercise. Using a randomly counterbalanced, crossover and double-blinded design, eleven male jumpers and sprinters (age: 18.7 ± 2.7 years) performed a half-squat exercise (4 x 12 repetitions at 70% of 1 RM), with eccentric action emphasized by using a flexible strip attached to their knees (Tirante Musculador®). They ingested either a capsule of placebo or caffeine (5 mg.kg-1 body mass) 24, 48 and 72 h after. Neuromuscular function and muscle power (vertical countermovement-jump test) were assessed before and after the half-squat exercise and 50 min after the placebo or caffeine ingestion at each time-point post-exercise. Sprint performance was measured at pre-test and 75 min after the placebo or caffeine ingestion at each time-point post-exercise. Maximal voluntary contraction (overall fatigue) and twitch torque (peripheral fatigue) reduced after the half-squat exercise (-11 and -28%, respectively, P < 0.05) but returned to baseline 24 h post-exercise (P > 0.05) and were not affected by caffeine ingestion (P > 0.05). The voluntary activation (central fatigue) and sprint performance were not altered throughout the experiment and were not different between caffeine and placebo. However, caffeine increased height and power during the vertical countermovement-jump test at 48 and 72 h post half-squat exercise, when compared to the placebo (P < 0.05). In conclusion, caffeine improves muscle power 48 and 72 h after an eccentric-based exercise, but it has no effect on neuromuscular function and sprint performance.

RCT Entities:   Population Interventions Outcomes

Introduction

Strength training with eccentric emphasis is used by sprinters and jumpers to provoke in-series sarcomere hypertrophy, which could increase the velocity of muscle contraction [1]. However, exercise with eccentric emphasis also increases muscle damage via increased sarcoplasmic rupture, dilation of the transverse tubule system, myofibrillar component distortion and sarcoplasmic reticulum fragmentation [2,3]. Due these structural changes, there is a late increase in creatine kinase (CK), a marker of muscle damage, with a concomitant increase in muscle pain after an eccentric exercise (24 to 72h post-exercise), the so-called delayed onset muscle soreness (DOMS). Increased muscle damage and DOMS might carry over into exacerbated neuromuscular fatigue, which can reduce performance during the subsequent training sessions even in individuals accustomed to this mode of training [4-6].

Only a few studies have investigated the time course of neuromuscular fatigue after eccentric exercise [4-8]. While it has consistently been demonstrated that DOMS, measured with a 10-point Pain Intensity Scale [9], peaks 48 h after an eccentric exercise, the time course of neuromuscular fatigue after an eccentric exercise is more controversial. Maximal voluntary contraction (MVC), an indicator of overall fatigue, can remain reduced from 24 h to 8 days or more after an eccentric exercise [4,8]. Central fatigue, measured by voluntary activation (VA) via the twitch interpolated technique is restored within 24 h to 72 h [4,6,10], while twitch torque (an indicator of peripheral fatigue) is restored in 24 h [10], 48 h [8] or sometimes more than 4 days [4,6]. Part of these inconsistencies might be due to different protocols inducing muscle damage such as elbow flexion [6], backward downhill walking [10] or heavy-resistance (strength) and jump training [8]. It is noteworthy that only one study recruited athletes as volunteers and used exercises that are part of the training routine of sprinters and jumpers [8]. Therefore, the time course of central and peripheral fatigue after an exercise with eccentric emphasis typically used in athlete’s training routine is underexplored in well-trained athletes and deserves further investigation.

Because athletes train almost every day, it is important to maintain performance during subsequent training sessions [11]. In this sense, caffeine (1,3,7-trimethylxanthine) seems to be a promising alternative. Following its removal from the World Anti-Doping Agency (WADA) Prohibited List in 2004, the use of caffeine for track and field athletes increased substantially [12]. The use of caffeine has been reported to be large in national/international level athletes (~48%) and in power athletes (~57%) (12). Because caffeine acts on the central nervous system (CNS) as an adenosine receptor antagonist, caffeine maintains neuronal excitability and counteracts central fatigue. Caffeine has also analgesic properties that could reduce the perception of pain during exercise [13]. In addition, caffeine improves excitation-contraction coupling by increasing calcium release/reuptake and Na+-K+-ATPase pump activity [14]. Caffeine also increases peak isokinetic torque after activities that resulted in exercise-induced muscle damage [15]. Considering these effects, caffeine might be an effective alternative to counteract the negative effect of DOMS provoked by a prior exercise with eccentric emphasis on power, sprint performance and central and peripheral fatigue.

Therefore, the first aim of the present study was to investigate the time course of CK, DOMS, jump and sprint performance and central and peripheral components of neuromuscular fatigue after a half-squat exercise with eccentric emphasis. The second aim was to investigate whether caffeine would influence central and peripheral components of neuromuscular fatigue and jump and sprint performance during the following days after the half-squat exercise with eccentric emphasis. We hypothesized that acute caffeine ingestion would improve central and peripheral components of neuromuscular fatigue and jump and sprint performance in the following days after a half-squat exercise with eccentric emphasis.

Materials and methods

Participants

Eleven well-trained young males’ sprinters and jumpers (age: 18.7 ± 2.7 years old; weight: 69.9 ± 6.4 kg; height: 180.7 ± 7.7 cm), accustomed to eccentric training and with low-to-moderate habitual caffeine consumption (< 80 mg.day-1) [16] participated in this study. The inclusion criteria were: (a) to be free from neuromuscular and musculoskeletal disorders; (b) to have had at least one year of experience with athletic training; (c) to have been training 5 to 6 times per week (12 to 15 hours per week) and; (d) to have been involved in national or international competitions in the year prior to the study. The participants and their guardians were informed about the procedures prior to the beginning of the study and signed a written informed consent. The research was conducted according to the Declaration of Helsinki and was approved by the Research Ethics Committee of the Federal University of Pernambuco.

Experimental design

The study was carried out within a 3-week period of a preparatory phase of an annual training periodization. In this phase, training sessions were designed to improve general resistance and not focused in eccentric training. Two preliminary sessions were designed to assess one-repetition maximal strength in a half-squat exercise (1RM) using a Tirante Musculador (see one-repetition maximal strength test section for details) and to familiarize the participants with the assessment of neuromuscular function. In the first preliminary session, anthropometric measurements were taken, and a full familiarization with the assessment of neuromuscular function and 1RM was performed. Forty-eight hours later, in the second preliminary session, 1RM was determined and participants had another opportunity to be familiarized with the assessment of neuromuscular function.

After two days of low-intensity training, premeasurements were performed over the next two days. On day 1, after a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching), pre-test sprints (one sprint of 30 m following by 5 min rest, one sprint of 50 m following by 7 min rest and one sprint of 100 m) were performed in an outdoor athletic track (Fig 1). On Day 2, baseline blood sample, DOMS, perceived recovery, neuromuscular function and vertical countermovement-jump test (CMJ) were assessed. Neuromuscular function was preceded by a warm-up (see neuromuscular function assessment section for details). Five minutes later, 4 sets of 12 repetitions at 70% of 1RM (2-min rest between sets) of a half-squat exercise with emphasis on eccentric action were performed using the Tirante Musculador. After exercise, the neuromuscular function and CMJ performance were reassessed. During the next three days (days 3, 4 and 5), a blood sample was collected and participants ingested a gelatin capsule containing either 5 mg.kg-1 body mass of anhydrous caffeine (CAF) or cellulose (PLA) 50 min before the assessment of DOMS, perceived recovery, neuromuscular function and CMJ. Participants also performed a sprint training session 75 min after the supplement ingestion.

Fig 1

Study timeline.

DOMS, Delayed Onset Muscle Soreness; PR, Perceived Recovery; NF, neuromuscular function; CMJ, countermovement jump; CAF, caffeine; PLA, placebo.

After a 7-day washout period performing a low-intensity training program, participants repeated the procedures described above, but those who had ingested caffeine in the first moment ingested a placebo and those who had ingested a placebo ingested caffeine (crossover design). The order of CAF and PLA ingestion were randomly counterbalanced and supplements offered in a double-blind manner. All tests were performed at the same time of day to avoid any effect of the circadian variation. Participants refrained from alcohol and caffeinated beverages throughout the study. Participants were also instructed to record their food and drink intake starting 48 h before the Day 1 until the Day 5 and then to replicate this intake in the second phase of the experiment. Participants were asked not to eat anything two hours before each test session.

One-repetition maximal strength test

The 1RM test was performed during a half-squat exercise with participants coupled to a Tirante Musculador (TMR-World, Barcelona, Spain). This accessory is a simple belt that allows anchor the calf and leave the body hanging with the gravity center far from the fulcrum of the knee and thus increasing the eccentric overload (Fig 2). The two flexible strips of the belt were attached below participants’ knees and fixed to a column. Participants performed the flexion and extension of the knees (90° amplitude). This flexible strip has been demonstrated suitable to increase eccentric action during a half-squat exercise [17]. Participants were familiar with this accessory as they had used it in their training routine as a form to increase eccentric action without using an excessive external load, which may reduce the risk of injuries associated with eccentric-based training, mainly in a preparatory phase of an annual training periodization [17]. The participants performed a warm up consisting of 15 repetitions at ~ 50% of the estimated 1RM [18]. After that, participants rested for 2 min and the load was increased to the expected 1 RM. Five attempts were necessaries to achieve the 1RM load (i.e., the maximum load that could be lifted once using the proper technique), with a 2 min interval between the attempts, as previously suggested [18]. The 1RM load was 130.2 ± 18.6 kg.

Fig 2

Half-squat exercise with eccentric emphasis performed with the Tirante Musculador.

A: Initial position of the movement; B: Final position of the movement.

Half-squat exercise with eccentric emphasis

Participants performed 15 repetitions of half-squat exercise at 50% of 1RM as warm up and after a 2 min rest, performed 4 sets of 12 repetitions at 70% of 1RM, with 2 min rest between sets. Each repetition took 3 s (2 seconds for the eccentric phase and 1 second for the concentric phase) and was controlled using a metronome. The eccentric action was emphasized by participants performing the half-squat using the Tirante Musculador, as detailed above. We ascertain this protocol with athetes’s coach to simulate their training routine. We chose a protocol able to provoke moderate rather than a more intense DOMS because athletes were in a preparatory phase of an annual training periodization; therefore, protocol to induced more intense DOMS would make athletes more susceptive to muscle injuries. The load at 70% of 1RM was 91.1 ± 13.9 kg.

Neuromuscular function assessment

The neuromuscular function was assessed with the participants seated on a custom-made bench with their hip and knees angles set at 120° and 90°, respectively. A non-compliant cuff attached to a calibrated linear strain gauge was fixed to the right ankle superior to the malleoli for force measurement (EMG System of Brazil, São Jose dos Campos, Brazil). A monopolar 0.5 cm diameter cathode electrode was positioned at the right femoral nerve for electrical stimulation (Ambu® Neuroline 715, Ballerup, Denmark). The anode was positioned on the gluteal fold opposite to the cathode. The position of the electrodes was marked with indelible ink to ensure identical placement in subsequent visits.

The optimal intensity of stimulation was determined by percutaneous electrical nerve stimuli (1 Hz and 80 μs duration) applied to the femoral nerve using an electrical stimulator (Neuro-TES, Neurosoft, Ivanovo, Russia). The intensity of stimulation began with 100 volts and increased 30 volts every 30 s until the occurrence of a plateau in the muscle membrane excitability (Mwave) and quadriceps twitch torque (Qtw). To ensure a supramaximal stimulus, the intensity of stimulation that Mwave and Qtw plateaued was further increased by 20%. The optimal intensity of stimulation was determined in the first visit and checked before every subsequent visit (301 ± 49 volts).

The neuromuscular function assessment (except immediately after the half-squat exercise) was preceded by a warm up composed of a 5 min jogging and 4 x 5 s isometric contractions of knee extension at ~60% of maximal subjective isometric force (30 s rest between contractions). The protocol of neuromuscular function assessment consisted of 6 x 5 s MVC of knee extensors, interspaced by 60-s recovery, as previously suggested [19]. Stimulus of 1 Hz (80 μs) was applied during each MVC (superimposed twitch) at the plateau of isometric force. Potentiated quadriceps twitch torque evoked by 1 Hz (Qtwpot) and paired pulses at 10 Hz (Qtw10) and 100 Hz (Qtw100) was measured 2, 4 and 6 s after each MVC, respectively. The Mwave peak-to-peak amplitude was calculated for each 1 Hz stimulus. A Qtw10·Qtw100−1 ratio was calculated to identify low-frequency fatigue [20]. The VA was measured by the following equation [21]: where superimposed is the difference between the force before the stimulus and the peak of force induced by the stimulus.

The average of the last four MVC for each time point was used for further statistical analysis of the neuromuscular function [19].

Countermovement jump test

Participants were familiar with CMJ in their training routine. The participants started in a standing position, dropped to a squatting position and jump upwards as high as possible. The jumps were performed with hands on the hip. The flying time during the CMJ was measured using a contact mat (Jump System Pro, Cefise, São Paulo, Brazil). The CMJ height was calculated from flying time, while CMJ power calculated from flying time and body mass, using a commercial software (Jump System 1.0, Cefise, São Paulo, Brazil). The jumps were performed three times (10 s interval) and the mean height and power used for further statistical analysis.

Sprint training

Sprint training was composed by a series of sprints similar to that performed in training routine. The sprint training session started with a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching). Then, participants performed: 1) 3 sets of 30-m sprints, with a 5 min rest between sets; 2) 3 sets of 50-m sprints, with a 7 min rest between sets and; 3) 3 sets of 100-m sprints, with a 10 min rest between sets. They were instructed to run as fast they could and wore the same footwear. Participants performed 10 min of running and stretching after training to cool down. The timing of the sprints was monitored using photocells (TC-System, Brower Timing System, US). Participants positioned immediately before the first photocell to start. The mean velocity in each distance was calculated and used for further analysis.

Blood sample

Blood samples (8 ml) were drawn by a professional phlebotomist from the antecubital vein by venipuncture and transferred to tubes containing Clot activator and gel for serum separation (SST II Plus, BD Vacutainer®, USA). The serum CK concentration was determined by an enzymatic method using commercial kits (Labtest Diagnostica S.A., Minas Gerais, Brazil), with the resultant reaction reading in a spectrophotometer (Genesys 10 S UV-vis, Thermo Electron Scientific Instruments, Madison, WI, United States).

Delayed onset muscle soreness and perceived recovery

The DOMS was measured with the Pain Intensity Scale ranging from 0 (no pain) to 10 (very intense pain, almost unbearable) [9]. The perception of recovery was measured on the Total Quality of Recovery Scale ranging from 6 to 20, where 6 is "nothing recovered" and 20 is "fully recovered" [22]. Participants were familiar with these scales as they had used them during their training routine.

Statistical analyses

Because of the lack of data regarding the caffeine effects on neuromuscular function post an eccentric-based exercise in athletes, the required sample size was estimated using an effect size (ES = 0.37) reported in a meta-analysis investigating the effect of caffeine ingestion on muscular strength [23]. With an alpha of 0.05 and a desired power of 0.80, the total sample size necessary to achieve statistical significance was estimated to be 10 participants. However, the starting sample size was increased to 11 participants, assuming that 10% might drop out during the data collection. The sample size calculation was performed using G*Power software (version 3.1.9.2, Kiel University, Germany).

The Shapiro-Wilk test was performed to determine the normality of the data. The neuromuscular function parameters (MVC, VA, Mwave ampl, Qtwpot, Qtw10, Qtw100 and Qtw10·Qtw100−1), CMJ, CK and perceived recovery were analyzed using a two-way, repeated-measures ANOVA [supplement (PLA and CAF) x time (baseline, post-exercise and 24, 48 and 72 h post exercise)], with a Duncan post-hoc test being utilized when ANOVA detected significant main effects and/or interaction. The pre-test sprint performance before the first and second experimental blocks were compared using a paired t test in order to check if athletes had fully recovered from the previous tests and training sessions. The performance during the sprint training was then analyzed separately using a two-way, repeated-measures ANOVA [supplement (PLA and CAF) x time (24, 48 and 72 h post exercise). The DOMS was analyzed using Friedman ANOVA followed by the Wilcoxon test because the DOMS had not shown a normal distribution. Partial eta squared (ηp2) was also calculated as a measure of the effect size and classified as small (ηp2 < .06), moderate (.06 ≤ ηp2 < .15) or large (ηp2 ≥ .15). The statistical significance was accepted at P < 0.05. Data are reported as mean ± SD, unless otherwise stated. Statistical analyses were performed using statistics package for Windows (version 10, StatSoft, Tulsa, OK, USA).

Results

Muscle damage, DOMS and perceived recovery

The CK increased above baseline levels 48 and 72 h post-exercise (main effect of time, F(4,32) = 10.810, P = 0.001, ηp2 = 0.57, Table 1). The DOMS increased from baseline to 24 h post-exercise, remaining above the baseline values until 72 h post-exercise (χ2(10) = 25.312, P = 0.001). Compared to baseline, the perceived recovery was always incomplete at 24, 48 and 72 h post-exercise (main effect of time, F(3, 27) = 12.429, P = 0.001, ηp2 = 0.58, Table 1). There was no effect of supplement for CK, DOMS and perceived recovery (P > 0.05).

Table 1

Creatine kinase, delayed onset muscle soreness and perceived recovery at baseline, and at 24, 48 and 72 h later a half-squat exercise with placebo or caffeine ingestion.

	PLACEBO				CAFFEINE
	Baseline	24 h	48 h	72 h	Baseline	24 h	48 h	72 h	F	p	ηp2
CK (U/L)	356.6 ± 220.1	381.9 ± 136.3	481.4 ± 66.3*	491.6 ± 209.8*	320.5 ± 148.5	337.9 ± 114.1	496.8 ±173.0*	497.3 ± 163.6*	1.07	0.38	0.11
	[199.1–514.1]	[284.3–479.4]	[331.3–631.4]	[326.1–657.1]	[214.2–426.7]	[256.2–419.5]	[373.0–620.5]	[371.5–623.1]
DOMS (score)	0.5 ± 1.0	2.3 ± 1.8*	1.9 ± 1.4*	3.0 ± 1.7*	0.9 ± 1.0	2.1 ± 1.8*	2.0 ± 1.5*	2.2 ± 1.4*		<0.01
PR (score)	17.4 ± 1.75	15.6 ± 1.74*	15.2 ± 2.49*	14.8 ± 2.63*	17.8 ± 1.66	16.2 ± 1.90*	14.7 ± 1.95*	15.0 ± 2.05*	1.02	0.39	0.10
	[16.2–18.6]	[14.4–16.8]	[13.5–16.9]	[13.0–16.5]	[16.7–18.9]	[14.9–17.5]	[13.4–16.0]	[13.5–16.4]

Values are expressed as mean ± SD [95% confidence interval], except for DOMS that values are expressed as median ± interquartile distance (non-normally distributed). CK, creatine kinase; DOMS, delayed onset muscle soreness; PR, perceived recovery.

*Significantly different from pre-exercise in both conditions (P<0.05).

Neuromuscular function

The MVC decreased significantly from baseline to immediate post-exercise (-11%) but recovered fully 24 h later (main effect of time, F(4,32) = 6.560, P = 0.001, ηp2 = 0.45, Fig 3A). Similarly, markers of peripheral fatigue (Qtwpot, Qtw10 and Qtw100) decreased significantly with the exercise (-30%, -35% and -12%, respectively) and recovered fully 24 h later (main effect of time, F(4,32) = 21.502, P = 0.001, ηp2 = 0.72, F(4,32) = 37.562, P = 0.001, ηp2 = 0.82 and F(4,32) = 17.928, P = 0.001, ηp2 = 0.69, Fig 3B, 3C and 3D, respectively). However, Qtw100 was slightly increased 48 and 72 h after the exercise in comparison to baseline (P = 0.001). The Qtw10·Qtw100−1 decreased significantly from baseline to immediate post-exercise (-25%) and recovered fully 24 h later (main effect of time, F(4,32) = 17.794, P = 0.001, ηp2 = 0.68, Fig 3E). The VA did not change throughout the experiment (main effect of time, F(4,24) = 2.297, P = 0.088, ηp2 = 0.27, Fig 3F). The Mwave amplitude increased significantly from baseline to post-exercise and returned to baseline values 24 h later (main effect of time, F(4,32) = 3.081, P = 0.029, ηp2 = 0.27, Fig 3G). Caffeine had no effect on any neuromuscular function parameters (P > 0.05).

Fig 3

Neuromuscular function assessed before and after a half-squat exercise, and at 24, 48 and 72 h after placebo or caffeine ingestion.

Values are expressed as mean ± SEM. A: MVC, maximal voluntary contraction; B: VA, voluntary activation; C: Mwave ampl, maximal M-wave amplitude; D: Qtwpot single stimulation (potentiated twitch); E: Qtw10 paired stimulations of 10 Hz; F: Qtw100 paired stimulations of 100Hz. G: Qtw10·Qtw100−1 ratio. *Significantly lower than pre, 24, 48 and 72 h post half-squat exercise under both conditions; **Significantly higher than pre- and post half-squat exercise under both conditions.

Countermovement jump and performance during the sprint training

There was a significant supplement vs. time interaction for CMJ performance. The height (F(4,28) = 3.299, P = 0.024, ηp2 = 0.32, Fig 4A) and power (F(4,28) = 2.885, P = 0.040, ηp2 = 0.29, Fig 4B) during the CMJ were higher 24 h after exercise compared to baseline and post-exercise in both supplements (P = 0.011 and P = 0.005, respectively). However, height and power during the CMJ 48 and 72 h after exercise remained above baseline values only when caffeine was ingested (P = 0.001). The height and power during the CMJ 48 and 72 h after exercise was also higher in caffeine compared to the placebo (P = 0.010).

Fig 4

Countermovement jump test performed before and after a half-squat exercise and at 24, 48 and 72 h after placebo or caffeine ingestion.

Values are expressed as mean ± SEM. A: Height; B: Power (relative to body mass). *Significantly higher than pre- and post half-squat exercise in both conditions; #Significantly higher than pre- and post half-squat exercise only in caffeine condition; †Significantly higher than placebo at the same time point.

Pre-test sprint performance did not differ between placebo and caffeine (30 m: 6.78 ± 0.24 vs. 6.72 ± 0.22 m.s-1, P = 0.372; 50 m: 7.48 ± 0.10 vs. 7.58 ± 0.06 m.s-1, P = 0.632; 100 m: 7.89 ± 0.15 vs. 7.98 ± 0.10 m.s-1, P = 0.392). In addition, sprint performance for all distances (30m, 50m and 100m) was not affected by supplement (F(1,9) = 0.083, P = 0.779, ηp2 = 0.009; F(1,9) = 0.203, P = 0.663, ηp2 = 0.022 and F(1,9) = 0.085, P = 0.776, ηp2 = 0.009, Table 2). However, for both conditions, sprint performance in 50 m was faster at 72 h compared to 24 h and 48 h after exercise (F(2,18) = 5.395, P = 0.014, ηp2 = 0.374) and faster in 100 m at 72 h compared to 48 h after exercise (F(2,18) = 6.367, P = 0.008, ηp2 = 0.414). There was no time effect for 30 m sprint (F(2,18) = 3.172, P = 0.066, ηp2 = 0.260).

Table 2

Sprint performance at 24, 48 and 72 h later a half-squat exercise with placebo or caffeine ingestion.

	PLACEBO			CAFFEINE
	24h	48h	72h	24h	48h	72h	F	p	ηp2
30m (m.sˉ¹)	6.56 ± 0.45	6.75 ± 0.39	6.65 ± 0.27	6.70 ± 0.42	6.78 ± 0.32	6.55 ± 0.37	1.29	0.29	0.12
	[6.25–6.86]	[6.48–7.01]	[6.46–6.83]	[6.41–6.97]	[6.56–7.00]	[6.28–6.82]
50m (m.sˉ¹)	7.49 ± 0.43	7.54 ± 0.55	7.26 ± 0.33*	7.42 ± 0.36	7.49 ± 0.38	7.27 ± 0.34*	0.36	0.70	0.03
	[7.19–7.78]	[7.16–7.91]	[7.04–7.48]	[7.17–7.67]	[7.23–7.74]	[7.02–7.51]
100m (m.sˉ¹)	7.89 ± 0.35	7.88 ± 0.38	7.81 ± 0.39**	7.85 ± 0.30	8.05 ± 0.30	7.80 ± 0.33**	1.71	0.20	0.15
	[7.64–8.12]	[7.61–8.14]	[7.55–8.07]	[7.56–8.14]	[7.75–8.34]	[7.44–8.14]

Values are expressed as mean ± SD [95% confidence interval].

*Significantly different from 24 h and 48 h after exercise in both conditions (P<0.05).

**Significantly different from 48 h after exercise in both conditions (P<0.05).

Discussion

The results of the present study indicate that although markers of muscle damage and DOMS (CK and pain feeling) remained elevated over 72 h after an exercise with eccentric emphasis, markers of central and peripheral fatigue returned to baseline values within 24 h. Caffeine did not affect neuromuscular function, but jump performance was improved with caffeine 48 and 72 h after an exercise with eccentric emphasis.

The CK serum concentration increased 48 h after exercise with eccentric emphasis and remained elevated until 72 h. However, DOMS increased 24 h after exercise with eccentric emphasis and remained above baseline levels until 72 h, which coincided with a reduction in the perception of recovery. The CK after an eccentric exercise peaks 1–4 days and can remain elevated for several days [3,24,25]. An important issue is that daily training results in persistent CK elevation in athletes, with resting values being higher than in non-athletes [25]. In the present study, even providing two days of low-intensity training before taking blood sample, we found baseline CK values ranging from 199 to 514 U/L, which are slightly above the upper reference limit established to general population (174 U/L) [26]. Because it is not possible to maintain athletes without any kind of training for days, a “true” CK baseline value is hard to find in athletes. However, the values found in the present study are in agreement with reference values proposed for male athletes (82–1083 U/L) [25]. We also found an increase in CK levels 48 h after exercise, regardless of the supplement ingested. This finding is in agreement with a study showing that CK peaks within 48 h after a marathon [24]. However, our CK level 48 h after the half-squat exercise was much lower than those reported 24 h after a marathon (434–844 U/L) [24]. This is expected because CK levels after prolonged exercise such as marathon can reach up to 50 times the rest values due to the greater muscle damage caused by this kind of activity [24].

The exercise did not influence central fatigue (VA), but induced to a peripheral fatigue, as evidenced by the large reduction in Qtwpot, Qtw10 and Qtw100 immediately post-exercise (-30, -35 and -12%, respectively) and a low-frequency fatigue, as evidenced by a reduction in Qtw10·Qtw100−1 (-25%). However, peripheral and low-frequency fatigue returned to baseline levels 24 h after the exercise. The time course of neuromuscular fatigue after a eccentric-based exercise is largely variable, with studies reporting that central fatigue returned to baseline within 24 to 96 h, while peripheral fatigue within 24 to 192 h [4-8,10]. Different protocols of eccentric exercise may take in account for these inconsistencies. In the present study, we optioned for an eccentric exercise nearer to that used in the athlete’s training routine. In addition, to avoid injuries, eccentric exercises used in regular training program are not designed to generate an elevated degree of muscle damage and DOMS. Our results suggest that although the considerable degree of peripheral fatigue after an exercise with eccentric emphasis (~ 30%), well-trained athletes accustomed to eccentric training can quickly restore their capacity to produce force (~24 h).

Caffeine had no influence on CK, DOMS or neuromuscular function. However, the height and power during the CMJ returned to baseline levels 48 h after the exercise with the placebo, while the height and power was maintained above baseline levels until 72 h after the exercise with caffeine. Muscle power was also higher in caffeine than in placebo at 48 and 72 h post-exercise. Previous studies have shown improved jump performance after the ingestion of 3 to 6 mg.kg-1 of caffeine [27-32]. Recent reviews concluded that caffeine promotes an ergogenic effect on muscle strength and power [23,33,34]. However, no study to date has demonstrated this improvement even after an exercise with eccentric emphasis using high performance sprinters and jumpers. Nevertheless, the improved power was not translated to an improved sprint performance in the present study. Similar findings have been reported showing no improvement in repeated-sprint ability with caffeine ingestion [30]. The shorter duration of the contraction and the simplicity of the technique may explain why performance was improved in the CMJ but not in the sprint with caffeine [30].

Our study demonstrated that a half-squat exercise session with an eccentric emphasis induces peripheral but not central fatigue, which is restored within 24 hours in well-trained jumpers and sprinters. Anhydrous caffeine (5 mg.kg-1) improved jump performance 48 and 72 h after a half-squat exercise with an emphasis on eccentric action. However, neuromuscular function and sprint performance were not influenced by caffeine intake. Our results has considerable practical relevance as they are indicating that caffeine can optimize jumping performance in well-trained athletes even when a certain degree of muscle damage and DOMS are present. Thus, ingestion of anhydrous caffeine in some jump training sessions may be an interesting strategy to improve training quality and performance of these athletes.

There are some limitations in the present study that should be mentioned. Our resistance training protocol generated mild to moderate muscle pain, resulting in no impairment of subsequent training capacity. Thus, whether caffeine would be useful when greater muscle pain is present deserves further investigation. Another potential limitation is that experimental conditions were performed only once each (one for placebo and another one to caffeine). Repetitions of the experimental blocks may have provided additional information regarding the reproducibility of our findings.

Conclusions

In conclusion, our study demonstrates that even with a certain low degree of muscle damage and DOMS over 72 h after an exercise with eccentric emphasis, neuromuscular function, muscle strength and sprint performance are preserved in well-trained sprinters and jumpers. Caffeine ingestion (5 mg.kg-1) improves muscle power 48 and 72 h after an exercise with eccentric emphasis, but it has no effect on neuromuscular function and sprint performance.

20 Aug 2019

PONE-D-19-16976

Effect of caffeine on neuromuscular function following eccentric-based exercise

PLOS ONE

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Reviewer #1: Manuscript Number: PONE-D-19-16976

Effect of Caffeine on neuromuscular function following eccentric-based exercise

General Comments

Authors carried out an interesting research regarding to the effect of caffeine on recovery of neuromuscular function, power and sprint performance following an eccentric-based exercises using a "Tirante Muscular� " in half squat exercise. Authors did a great job controlling many variables providing a broad vision when studying central and peripheral fatigue, DOMS, perceived recovery, muscle damage, CMJ and Sprint. However, the manuscript is well written and very good organized. Should be highlight why the Tirante Muscular� has been used, since it is not clear the reason that you chose this tool and not a Smith Machine or free weight. On the other hand, in the paragraph of experimental design, authors describe the sprint pre-test as “one sprint of 30 m, one of 50 m and one of 100m”. In the post- test, has not been carried out the same procedure, conversely have been performed: “1) 3 sets of 30m sprints, with a 5min rest between sets; 2) 3 sets of 50 m sprints, with a 7 min rest between sets and; 3) 3 sets of 100 m sprints, with a 10 min rest between sets”. These two protocols are different and the results cannot be compared due to the volume and the fatigue in each test. It is a limitation of this study that should be shown in the manuscript. It is possible to think that the performance in the sprint has not improved due to the fatigue that the post-test supposes. Perhaps a single series of 30m would have been enough to compare the performance in the sprint effectively. By this is important that authors explain it. Finally, only some changes I can propose to improve this manuscript. Thus, I considered that the paper could be accepted if the authors could correct mistakes pointed out. My specific comments are presented below.

Specific Comments

Page 2, line 42: Before “and”, “,” should not be used. On the other hand, between “72” and “h” the “-” it is not necessary and the authors due write in the same form along the manuscript because in some case don’t use hyphen.

Page 2, line 45: The use of concept baseline is not appropriated to refer to Sprint performance. This concept is well used to muscle damage, DOMS or RT, but sprint need a warm up and activation process to do the best performance. By this, change “Baseline” by “Pre-test” could be more appropriated.

Page 2, line 48,52: Remove the hyphen (24 h) (48 h) (72 h).

Page 3, line 73: It would be good to write a brief description of how DOMS is measured in the literature.

Page 3, line 73,77,78: Remove the hyphen (24 h) (48 h) (72 h).

Page 4, line 101: It is necessary write the name of Creatine Kinase before (CK) since is the first time that appear this concept during de manuscript.

Page 5, line 111-112: After “years old” should be used colon not comma and it is necessary write “height:” before 180.7…. Also, it is interesting to report the Mean and SD of 1RM load or the load used corresponding with 70%1RM.

Page 5, line 123: The sentence starts very similar like the sentence before. Change “study” by “research” could be a good strategy of avoid be repetitive.

Page 5, line 126: Explain if the 1RM test was carried out with Tirante Muscular� .

Page 6, line 133-134: Unify the way to refer days (with numbers “2” or letters “two”).

Page 6, line 134: Change “baseline” by “Pre”. Also, before “and”, “,” should not be used, remove it.

Page 6, line 134-135: How much time was used to recover between the sprints?

Page 6, line 134-137: Was carried out some warm up before Sprints and CMJ? Could you explain it?

Page 6, line 139: Explain if the half-squat exercise was performed with Tirante Muscular�.

Page 6, line 157: Remove the hyphen (48 h).

Page7, line 173,175: Remove the hyphen (2 min).

Page 8, line 182,183: Remove the hyphens (2 min) (3 s).

Page 8, line 193: Remove the hyphen (0.5 min).

Page 8, line 202: Remove the hyphen (30 s).

Page 9, line 209,211,212,216: Remove the hyphens.

Page 10, line 240: Could you explain the stretching exercises that was carried out? It is known that passive stretching can impair the performance.

Page 11, line 251: Explain if a nurse or another qualified professional took the serum extractions.

Page 11, line 252: You have used “Creatine Kinase” before, the abbreviation is enough in this case.

Page 11, line 258, 260: Unify the way to refer the numbers of the scale (with numbers “0” or letters “zero” and “six”). Change words by numbers.

Page 12, line 284,285: Before “and”, “,” should not be used, remove it.

Page 12, line 289: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.

Page 13, line 299: Before “and”, “,” should not be used, remove it.

Page 13, line 312: Add “exercise” after a half squat.

Page 14, line 333: You can use “CMJ” instead of “countermovement jump”

Page 14, line 334: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.

Page 15, line 346: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.

Page 16, line 368: Add “s” after “athletes’ ”

Page 16, line 378: Remove the hyphens (48 h) and (72 h)

Page 17, line 392: It would be interesting to complete the paragraph with a section of limitations, perhaps report about the pre and post sprints test or regarding to only use one caffeine intake protocol.

Figures 3 and 4: Unify the levels of values in the Y axis of all figures. MVC has nine levels, Qtwpost has seven levels or Qtw100 has eight levels.

Reviewer #2: Review to: PONE-D-19-16976; Effect of caffeine on neuromuscular function following eccentric-based exercise.

Major comments.

This experiment was designed to assess the neuromuscular effects of caffeine intake during the recovery phase of an eccentric-based exercise protocol that induces muscle damage. Overall, the experiment seems well designed, it contains several measurements to fulfill the objectives of the investigation and it is conveniently described in a well-written manuscript. However, the experiment has a serious flaw: it is designed to assess caffeine’s effects in the recovery phase of a muscle-damaging exercise when most, if not all, performance variables returned to basal -non-fatigued- values within 24 hours after the end of exercise. This fact is likely due to the use of a “light” eccentric exercise protocol that produced very low levels of muscle damage – a pilot study would have been helpful to detect the inefficacy of this protocol to produce moderate muscle damage”.

Thus, although I found merit in the experiment and I congratulate authors for the hard work developed for this investigation, I would suggest that authors reconsider the use of “recovery” through the manuscript because this is not what they were investigating -the recovery lasted < 24 h and they did not perform any measurement in this time. For example, the conclusion of the abstract should indicate that:

“Caffeine improves muscle power 48 and 72 hours after an eccentric-based exercise…”.

A limitation paragraph including this aspect is highly recommended.

Other comments.

Introduce information about the use of caffeine in athletics. Is this common?

Indicate why you selected this protocol of eccentric exercise. And discuss the lack of a relevant increase in CK (for instance, you could compare these values with the ones found after a marathon).

Indicate the habituation to caffeine of these individuals.

The CK concentrations before exercise seems quite high (especially one participant with > 500 U/L). Please, discuss this in the light of previous investigations. Did they have previous muscle damage due to training?

Why there is no data on jump height?

Please, indicate the calculation of the sample size or at least, the statistical power with this sample of 11 individuals.

A paragraph of practical applications for jumpers and sprinter might be suitable. How they should use caffeine during their training. Before all sessions? 48 h after a high-intensity session?

Line 397. “sprinters” is twice

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Reviewer #1: Yes: González-Hernández Jorge Miguel

Reviewer #2: No

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Submitted filename: REVISION.docx

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Submitted filename: PONE REVIEW.docx

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19 Sep 2019

September 16, 2019

Dear Dr. Daniel Boullosa

Academic Editor of the PLOS ONE

We thank the referees for their time and effort spent in carefully reviewing our manuscript and providing such valuable comments. We also thank the editor for the opportunity to revise our manuscript: PONE-D-19-16976,

“Effect of caffeine on neuromuscular function following eccentric-based exercise”. We carefully considered all referees’ suggestions and included corresponding alterations in the manuscript. Revised points in the manuscript are highlighted in yellow and a point-by-point reply is detailed below. We believe all these suggestions have improved the quality of our manuscript.

Reviewer #1

Authors did a great job controlling many variables providing a broad vision when studying central and peripheral fatigue, DOMS, perceived recovery, muscle damage, CMJ and Sprint. However, the manuscript is well written and very good organized. Should be highlight why the Tirante Muscular� has been used, since it is not clear the reason that you chose this tool and not a Smith Machine or free weight. On the other hand, in the paragraph of experimental design, authors describe the sprint pre-test as “one sprint of 30 m, one of 50 m and one of 100m”. In the post- test, has not been carried out the same procedure, conversely have been performed: “1) 3 sets of 30m sprints, with a 5min rest between sets; 2) 3 sets of 50 m sprints, with a 7 min rest between sets and; 3) 3 sets of 100 m sprints, with a 10 min rest between sets”. These two protocols are different and the results cannot be compared due to the volume and the fatigue in each test. It is a limitation of this study that should be shown in the manuscript. It is possible to think that the performance in the sprint has not improved due to the fatigue that the post-test supposes. Perhaps a single series of 30m would have been enough to compare the performance in the sprint effectively. By this is important that authors explain it.

Response: Thank you for these positive comments. We appreciate your suggestions to improve our paper. Regarding the Tirante Muscular®, we have chosen this device to increase the eccentric action during the half-squat exercise. This device is largely used by athletes in their training routine because they do not need to use excessive high external loads to increase eccentric action, avoiding injuries during eccentric-based training. A previous study showed that eccentric action is considerably increased by using this device (EDIR DA SILVA et al., 2005; http://www.redalyc.org/articulo.oa?id=551656963007). We have added this information in the manuscript on Page 8, Line 179 as follow:

“This flexible strip has been demonstrated suitable to increase eccentric action during a half-squat exercise (17). Participants were familiar with this accessory as they had used it in their training routine as a form to increase eccentric action without using an excessive external load, which may reduce the risk of injuries associated with eccentric-based training, mainly in a preparatory phase of an annual training periodization (17)”.

Regarding the sprint training performance, we agree that pre-test cannot be directly compared to 24h, 48h and 72h recovery. We performed the pre-test sprints to check whether athletes would be fully recovered at the transition from the first experimental block (using placebo or caffeine) to the second experimental block (when supplement was inverted). Only one repetition of each distance was performed to avoid the development of fatigue and to not influence the following days tests. This procedure guaranteed that all blocks (placebo and caffeine) started with participants having fully recovered from previous tests and training and with the same recovery level in both conditions. For the post-test (i.e., sprints at 24, 48 and 72h), more series were performed to simulate a typical sprint training session and to understand whether caffeine would be helpful to improve sprint training performance. We agree these assumptions were not clear in the paper and our statistical analysis might have not fit well to this purpose. Thus, in this new version we have compared the two pretests sprint performance just to check if there was difference in the recovery from an experimental block to another, and then separately compared the sprint performance between placebo and caffeine over the posttest period. We have included this information on Page 13, Line 294: “The pre-test sprint performance before the first and second experimental blocks were compared using a paired t test in order to check if athletes had fully recovered from the previous tests and training sessions. The performance during the sprint training was then analyzed separately using a two-way, repeated-measures ANOVA [supplement (PLA and CAF) x time (24, 48 and 72 h post exercise)”.

Page 16, Line 362 “Pre-test sprint performance did not differ between placebo and caffeine (30 m: 6.78 ± 0.24 vs. 6.72 ± 0.22 m.s-1, P = 0.372; 50 m: 7.48 ± 0.10 vs. 7.58 ± 0.06 m.s-1, P = 0.632; 100 m: 7.89 ± 0.15 vs. 7.98 ± 0.10 m.s-1, P = 0.392). In addition, sprint performance for all distances (30m, 50m and 100m) was not affected by supplement (F(1,9) = 0.083, P = 0.779 , ηp2 = 0.009; F(1,9) = 0.203 , P = 0.663, ηp2 = 0.022 and F(1,9) = 0.085, P = 0.776 , ηp2 = 0.009, Table 2). However, for both conditions, sprint performance in 50 m was faster at 72 h compared to 24 h and 48 h after exercise (F(2,18) = 5.395 , P = 0.014, ηp2 = 0.374) and faster in 100 m at 72 h compared to 48 h after exercise (F(2,18) = 6.367, P = 0.008 , ηp2 = 0.414). There was no time effect for 30 m sprint (F(2,18) = 3.172, P = 0.066 , ηp2 = 0.260)”.

Minor:

Page 2, line 42: Before “and”, “,” should not be used. On the other hand, between “72” and “h” the “-” it is not necessary and the authors due write in the same form along the manuscript because in some case don’t use hyphen.

Response: Thank you for this recommendation. We have modified accordingly throughout the manuscript.

Page 2, line 45: The use of concept baseline is not appropriated to refer to Sprint performance. This concept is well used to muscle damage, DOMS or RT, but sprint need a warm up and activation process to do the best performance. By this, change “Baseline” by “Pre-test” could be more appropriated.

Response: We have changed “baseline” for “pre-test” when referring to sprint throughout the text, as suggested.

Page 2, line 48,52: Remove the hyphen (24 h) (48 h) (72 h).

Response: We have removed accordingly (Page 2, Line 48, 52).

Page 3, line 73: It would be good to write a brief description of how DOMS is measured in the literature.

Response: This has been added on Page 12, Line 273 “The DOMS was measured with the Pain Intensity Scale ranging from 0 (no pain) to 10 (very intense pain, almost unbearable) (9)”.

Page 3, line 73,77,78: Remove the hyphen (24 h) (48 h) (72 h).

Response: We have removed accordingly (Page 3, Line 74, 77, 79, 80).

Page 4, line 101: It is necessary write the name of Creatine Kinase before (CK) since is the first time that appear this concept during de manuscript.

Response: We thank the reviewer to pick up this mistake. The CK has been defined now in his first apparition “… creatine kinase (CK), a marker of muscle damage”. (Page 3, line 66).

Page 5, line 111-112: After “years old” should be used colon not comma and it is necessary write “height:” before 180.7…. Also, it is interesting to report the Mean and SD of 1RM load or the load used corresponding with 70%1RM.

Response: We thank the reviewer to pick up these typo error. This part of the text is now reading “…(age: 18.7 ± 2.7 years old; weight: 69.9 ± 6.4 kg; height: 180.7 ± 7.7 cm)”. (Page 5, line 115-116).

The 1RM load and the load corresponding to 70% of 1 RM were also inserted:

“The 1RM load was 130.2 ± 18.6 kg”. (Page 8, line 188).

“The load at 70% of 1RM was 91.1 ± 13.9 kg”. (Page 9, line 204).

Page 5, line 123: The sentence starts very similar like the sentence before. Change “study” by “research” could be a good strategy of avoid be repetitive.

Response: The first sentence has been changed to “(The research…)” (Page 5, line 123).

Page 5, line 126: Explain if the 1RM test was carried out with Tirante Muscular� .

Response: We have included this information accordingly “Two preliminary sessions were designed to assess one-repetition maximal strength in a half-squat exercise (1RM) using a Tirante Musculador® (see one-repetition maximal strength test section for details)”. (Page 5, line 130-132).

Page 6, line 133-134: Unify the way to refer days (with numbers “2” or letters “two”).

Response: This sentence has been changed to “After two days of low-intensity training, premeasurements were performed over the next two days”. (Page 6, line 138-139).

Page 6, line 134: Change “baseline” by “Pre”. Also, before “and”, “,” should not be used, remove it.

Response: We have modified accordingly. This sentence has been changed to “premeasurements were performed…”. (Page 6, line 138).

Page 6, line 134-135: How much time was used to recover between the sprints?

Response: We have added this information “pre-test sprints (one sprint of 30 m following by 5 min rest, one sprint of 50 m following by 7 min rest and one sprint of 100 m)”. (Page 6, line 140-141).

Page 6, line 134-137: Was carried out some warm up before Sprints and CMJ? Could you explain it?

Response: Athletes performed a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching) before sprints. This information has been included on Page 6, Line 139 “On day 1, after a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching)”. Because athletes performed the CMJ immediately after the neuromuscular function assessment, and neuromuscular assessment was precluded by a warm-up, athletes were already warmed up and no additional warm-up was required before CMJ. Details of the warm-up before neuromuscular function assessment is provided on Page 6, Line 144 “Neuromuscular function was preceded by a warm-up (see neuromuscular function assessment section for details)”.

Page 9, Line 224-227 “The neuromuscular function assessment (except immediately after the half-squat exercise) was preceded by a warm up composed of a 5 min jogging and 4 x 5 s isometric contractions of knee extension at ~60% of maximal subjective isometric force (30 s rest between contractions)”.

Page 6, line 139: Explain if the half-squat exercise was performed with Tirante Muscular�.

Response: We have included accordingly “a half-squat exercise with emphasis on eccentric action were performed using the Tirante Musculador®”. (Page 6, line 146-147).

Page 6, line 157: Remove the hyphen (48 h).

Response: This has been removed accordingly (Page 7, line 167).

Page7, line 173,175: Remove the hyphen (2 min).

Response: This has been removed accordingly (Page 8, line 185, 187).

Page 8, line 182,183: Remove the hyphens (2 min) (3 s).

Response: This has been removed accordingly (Page 8, line 196,197).

Page 8, line 193: Remove the hyphen (0.5 min).

Response: This has been removed accordingly (Page 9, line 211).

Page 8, line 202: Remove the hyphen (30 s).

Response: This has been removed accordingly (Page 9, line 219).

Page 9, line 209,211,212,216: Remove the hyphens.

Response: This has been removed accordingly (Page 9-10, line 225, 226, 228, 231).

Page 10, line 240: Could you explain the stretching exercises that was carried out? It is known that passive stretching can impair the performance.

Response: We were aware that passive stretching impairs sprint performance; therefore, athletes performed dynamic stretching exercises, as they usually do before their training and competition. Dynamic stretching is recommended as warm up for sprint due to the similarity of the movement patterns, the consequent increase in body temperature and proprioception aid (WINCHESTER et al., 2008; doi: 10.1519/JSC.0b013e31815ef202). We have added that dynamic stretching was used as warm up “The sprint training session started with a 20 min warm-up (10 minutes running + 10 minutes of dynamic stretching)”. (Page 11, line 253).

Page 11, line 251: Explain if a nurse or another qualified professional took the serum extractions.

Response: All blood samples were collected by an accredited professional phlebotomist. We have included this information “Blood samples (8 ml) were drawn by a professional phlebotomist from the antecubital vein by venipuncture…”. (Page 11, line 264).

Page 11, line 252: You have used “Creatine Kinase” before, the abbreviation is enough in this case.

Response: We thank the reviewer to pick up this mistake. The sentence now reads “The serum CK concentration was determined…”. (Page 11, line 266).

Page 11, line 258, 260: Unify the way to refer the numbers of the scale (with numbers “0” or letters “zero” and “six”). Change words by numbers.

Response: This has been changed accordingly “The DOMS was measured with the Pain Intensity Scale ranging from 0 (no pain) to 10 (very intense pain, almost unbearable) (9). The perception of recovery was measured on the Total Quality of Recovery Scale ranging from 6 to 20, where 6 is "nothing recovered" and 20 is "fully recovered”. (Page 12, line 273-276).

Page 12, line 284,285: Before “and”, “,” should not be used, remove it.

Response: We have removed accordingly (Page 12, line 290,291).

Page 12, line 289: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.

Response: The sentence now reads “Creatine kinase, delayed onset muscle soreness and perceived recovery at baseline, and at 24, 48 and 72 h later a half-squat exercise” . (Page 14, line 316).

Page 13, line 299: Before “and”, “,” should not be used, remove it.

Response: We have removed accordingly (Page 14, line 329).

Page 13, line 312: Add “exercise” after a half squat.

Response: We have added accordingly. The text now reads “Neuromuscular function assessed before and after a half-squat exercise”. (Page 15, line 341).

Page 14, line 333: You can use “CMJ” instead of “countermovement jump”

Response: The sentence now reads “Countermovement jump test performed before and after a half-squat exercise”. (Page 16, line 372).

Page 14, line 334: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.

Response: We have changed accordingly. This text now reads “. Countermovement jump test performed before and after a half-squat exercise”. (Page 16, line 372).

Page 15, line 346: If you have used “a half-squat exercise” during all manuscript in this case is more appropriate than “a half-squat resistance training”. Change it.

Response: We have changed accordingly. This text now reads. “Sprint performance at 24, 48 and 72 h later a half-squat exercise”. (Page 17, line 382).

Page 16, line 368: Add “s” after “athletes’ ”

Response: We have modified accordingly (Page 18, line 422).

Page 16, line 378: Remove the hyphens (48 h) and (72 h)

Response: This has been removed (Page 19, line 432).

Page 17, line 392: It would be interesting to complete the paragraph with a section of limitations, perhaps report about the pre and post sprints test or regarding to only use one caffeine intake protocol.

Response: A new paragraph has been added to address limitations. We have added the low level of pain generated by our resistance training protocol (suggestion of reviewer 2) and using only one caffeine intake protocol (your suggestion) as limitations. However, based in your first comment, we have reanalyzed the sprint data and felt we have solved the problem of the differences in the numbers of sprints between pre and post sprints (please, see our answer for your first comment). Thus, we think this is now not properly a limitation. The limitation paragraph reads (Page 20, line 452-458) “There are some limitations in the present study that should be mentioned. Our resistance training protocol generated mild to moderate muscle pain, resulting in no impairment of subsequent training capacity. Thus, whether caffeine would be useful when greater muscle pain is present deserves further investigation. Another potential limitation is that experimental conditions were performed only once each (one for placebo and another one to caffeine). Repetitions of the experimental blocks may have provided additional information regarding the reproducibility of our findings”.

Figures 3 and 4: Unify the levels of values in the Y axis of all figures. MVC has nine levels, Qtwpost has seven levels or Qtw100 has eight levels.

Response: We have modified Y- axis in figures 3 and 4 and now all Y-axis have nine levels.

Reviewer #2:

This experiment was designed to assess the neuromuscular effects of caffeine intake during the recovery phase of an eccentric-based exercise protocol that induces muscle damage. Overall, the experiment seems well designed, it contains several measurements to fulfill the objectives of the investigation and it is conveniently described in a well-written manuscript. However, the experiment has a serious flaw: it is designed to assess caffeine’s effects in the recovery phase of a muscle-damaging exercise when most, if not all, performance variables returned to basal -non-fatigued- values within 24 hours after the end of exercise. This fact is likely due to the use of a “light” eccentric exercise protocol that produced very low levels of muscle damage – a pilot study would have been helpful to detect the inefficacy of this protocol to produce moderate muscle damage”. Thus, although I found merit in the experiment and I congratulate authors for the hard work developed for this investigation, I would suggest that authors reconsider the use of “recovery” through the manuscript because this is not what they were investigating -the recovery lasted < 24 h and they did not perform any measurement in this time. For example, the conclusion of the abstract should indicate that: “Caffeine improves muscle power 48 and 72 hours after an eccentric-based exercise…”. A limitation paragraph including this aspect is highly recommended.

Response: We are happy that you have appreciated our experiment design and the quality of writing of the paper. We also thank you for your suggestions to improve our paper. We agree that our eccentric exercise protocol was not intense enough to provoke long impairment in our main outcomes. A pilot study would be helpful, but as we recruited athletes involved in national and international competitions and they were engaged in experiments taking 3 weeks, we were afraid to propose a longer experimental design and impair their preparation and carry to decline participation. In addition, we were afraid to propose a more intense protocol to produce larger muscle damage because they were in the preparatory phase of an annual training periodization; therefore, more susceptive to muscle injuries if using intense muscle damage protocol. To not miss the opportunity to test performance in athletes of this level, we ascertain this light protocol with their coach. However, we recognize this is a limitation and the term “recovery” should not be used. Based on your suggestion, we have removed the use of recovery throughout the manuscript and replaced to “after a half-squat exercise with eccentric emphasis” or something similar. Conclusion in the abstract was also altered as suggested (Page 2, Line 54) to “In conclusion, caffeine improves muscle power 48 and 72 h after an eccentric-based exercise, but it has no effect on neuromuscular function and sprint performance”. We have also added a paragraph in the discussion recognized this point as a limitation, as suggested (Page 20, line 452-458) “There are some limitations in the present study that should be mentioned. Our resistance training protocol generated mild to moderate muscle pain, resulting in no impairment of subsequent training capacity. Thus, whether caffeine would be useful when greater muscle pain is present deserves further investigation. Another potential limitation is that experimental conditions were performed only once each (one for placebo and another one to caffeine). Repetitions of the experimental blocks may have provided additional information regarding the reproducibility of our findings”.

Based on the data presented, I have the following comments/questions.

1. Introduce information about the use of caffeine in athletics. Is this common?

Response: Thank you for this question. Yes, it was. Since caffeine has been removed from the banned list of substances of World Anti-Doping Agency (WADA), there are a progressive increase in caffeine use as an ergogenic resource by athletes of various sports, including athletics (CHESTER & WOJEK, 2008; DOI 10.1055/s-2007-989231). We have included a sentence in the introduction providing this information (Page 4, line 90-93) “Following its removal from the World Anti-Doping Agency (WADA) Prohibited List in 2004, the use of caffeine for track and field athletes increased substantially (12). The use of caffeine has been reported to be large in national/international level athletes (~48%) and in power athletes (~57%) (12)”.

2. Indicate why you selected this protocol of eccentric exercise. And discuss the lack of a relevant increase in CK (for instance, you could compare these values with the ones found after a marathon).

Response: We have included an explanation about this choice on Page 8, Line 200-204 “We ascertain this protocol with athetes’s coach to simulate their training routine. We chose a protocol able to provoke moderate rather than a more intense DOMS because athletes were in a preparatory phase of an annual training periodization; therefore, protocol to induced more intense DOMS would make athletes more susceptive to muscle injuries”.

We have also included in the discussion section the lack of a relevant increase in CK and the comparison with values after a marathon, as suggested (Page 18, Line 406- 412) “We also found an increase in CK levels 48 h after exercise, regardless of the supplement ingested. This finding is in agreement with a study showing that CK peaks within 48 h after a marathon (24). However, our CK level 48 h after the half-squat exercise was much lower than those reported 24 h after a marathon (434-844 U/L) (24). This is expected because CK levels after prolonged exercise such as marathon can reach up to 50 times the rest values due to the greater muscle damage caused by this kind of activity (24)”.

3. Indicate the habituation to caffeine of these individuals.

Response: We have included the athletes’ daily caffeine consumption on Page 5, Line 117 “low-to-moderate habitual caffeine consumption (< 80 mg.day-1) (16)”.

4. The CK concentrations before exercise seems quite high (especially one participant with > 500 U/L). Please, discuss this in the light of previous investigations. Did they have previous muscle damage due to training?

Response: Thank you for this question. The baseline CK upper limit for general population is 174 U/L. We found an average baseline value of 330 U/L, which suggests a slightly already elevated CK. Unfortunately, daily training results in persistent CK elevation in athletes, which makes hard to find a “true” baseline. We tried to control this by providing two days of low-intensity training before blood sample, as it would be difficult to maintain high performance athletes without any kind of training. Although we have not got values below 174 U/L, the values were quite close (~ 330 U/L) and not different between supplements, which suggests that any effect of previous training on CK as similar across experimental conditions. We have added a discussion about this on Page 17, Line 397-406 “The CK after an eccentric exercise peaks 1–4 days and can remain elevated for several days (3,24,25). An important issue is that daily training results in persistent CK elevation in athletes, with resting values being higher than in non-athletes (25). In the present study, even providing two days of low-intensity training before taking blood sample, we found baseline CK values ranging from 199 to 514 U/L, which are slightly above the upper reference limit established to general population (174 U/L) (26). Because it is not possible to maintain athletes without any kind of training for days, a “true” CK baseline value is hard to find in athletes. However, the values found in the present study are in agreement with reference values proposed for male athletes (82-1083 U/L) (25)”.

5. Why there is no data on jump height?

Response: We have included data from jump height in this new version, as suggested. We have changed information in Methods section (Page 10, Line 246) “The CMJ height was calculated from flying time, while CMJ power calculated from flying time and body mass, using a commercial software (Jump System 1.0, Cefise, São Paulo, Brazil).” and Results section (Page 15, Line 353-360) “There was a significant supplement vs. time interaction for CMJ performance. The height (F(4,28) = 3.299, P = 0.024, ηp2 = 0.32, Fig 4A) and power (F(4,28) = 2.903, P = 0.039, ηp2 = 0.29, Fig 4B) during the CMJ were higher 24 h after exercise compared to baseline and post-exercise in both supplements (P = 0.011 and P = 0.005, respectively). However, height and power during the CMJ 48 and 72 h after exercise remained above baseline values only when caffeine was ingested (P = 0.001). The height and power during the CMJ 48 and 72 h after exercise was also higher in caffeine compared to the placebo (P = 0.010)”. We have also included CMJ height in figure 4.

6. Please, indicate the calculation of the sample size or at least, the statistical power with this sample of 11 individuals.

Response: We have added a paragraph in the statistical analysis section explaining the calculation of the sample size “Because of the lack of data regarding the caffeine effects on neuromuscular function post an eccentric-based exercise in athletes, the required sample size was estimated using an effect size (ES = 0.37) reported in a meta-analysis investigating the effect of caffeine ingestion on muscular strength (23). With an alpha of 0.05 and a desired power of 0.80, the total sample size necessary to achieve statistical significance was estimated to be 10 participants. However, the starting sample size was increased to 11 participants, assuming that 10% might drop out during the data collection. The sample size calculation was performed using G*Power software (version 3.1.9.2, Kiel University, Germany). (Page 12, Line 280-288).

7. A paragraph of practical applications for jumpers and sprinter might be suitable. How they should use caffeine during their training. Before all sessions? 48 h after a high-intensity session?

Response: Thank you four this suggestion. A new paragraph has been added to address practical applications (Page 19, line 447-451) “Our results has considerable practical relevance as they are indicating that caffeine can optimize jumping performance in well-trained athletes even when a certain degree of muscle damage and DOMS are present. Thus, ingestion of anhydrous caffeine in some jump training sessions may be an interesting strategy to improve training quality and performance of these athletes”.

8. Line 397. “sprinters” is twice.

Response: This typo error has been fixed accordingly. The text now reads “(sprinters and jumpers)”. (Page 20, line 464).

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23 Oct 2019

Effect of caffeine on neuromuscular function following eccentric-based exercise

PONE-D-19-16976R1

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Reviewer #1: The authors have done a good job of completing the manuscript with the proposed comments. Well done!

Reviewer #2: The authors have done a commendable work in improving the manuscript. In this new version, they have changed the focus of their investigation as suggested (from “muscle damage” to muscle performance after eccentric-based exercise), have discussed variables not included in the previous versions (i.e., jumps, CK concentrations) and have included limitations according to the weakness of their experimental design.

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Reviewer #1: Yes: González-Hernández Jorge Miguel

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28 Oct 2019

PONE-D-19-16976R1

Effect of caffeine on neuromuscular function following eccentric-based exercise

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