Dietary and Biological Assessment of the Omega-3 Status of Collegiate Athletes: A Cross-Sectional Analysis.

Omega-3 fatty acids (ω-3 FA) are associated with cardiovascular health, brain function, reduction of inflammation, and several other physiological roles of importance to competitive athletes. The ω-3 FA status of National Collegiate Athletic Association (NCAA) Division I athletes has not been well-described. The purpose of this study was to evaluate the ω-3 FA status of NCAA Division I athletes using dietary and biological assessment methodology. Athletes from nine NCAA Division I institutions from throughout the U.S. (n = 1,528, 51% male, 34 sports represented, 19.9 ± 1.4 years of age) completed a food frequency questionnaire (FFQ) to assess ω-3 FA from diet and supplements. Omega-3 Index (O3i) was evaluated in a sub-set of these participants (n = 298, 55% male, 21 sports represented, 20.0 ± 1.3 years of age) using dried blood spot sampling. Only 6% (n = 93) of athletes achieved the Academy of Nutrition & Dietetics' recommendation to consume 500 mg DHA+EPA per day. Use of ω-3 FA supplements was reported by 15% (n = 229) of participants. O3i was 4.33 ± 0.81%, with no participants meeting the O3i benchmark of 8% associated with the lowest risk of cardiovascular disease. Every additional weekly serving of fish or seafood was associated with an absolute O3i increase of 0.27%. Overall, sub-optimal ω-3 FA status was observed among a large, geographically diverse group of male and female NCAA Division I athletes. These findings may inform interventions aimed at improving ω-3 FA status of collegiate athletes. Further research on athlete-specific ω-3 FA requirements is needed.

Introduction

Omega-3 polyunsaturated fatty acids (ω-3 FA), namely long-chain eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), serve as structural components within phospholipid cell membranes. These ω-3 FA have also been shown to play important physiological roles among the cardiovascular [1-6], nervous [7-13], and skeletal muscle systems [14-18], and in the body’s inflammatory response [19-26]. In athletes, ω-3 FA have been associated with the management of exercise-induced oxidative stress [19, 20, 23–25], delayed onset muscle soreness [21, 22, 25, 26], oxygen efficiency during aerobic exercise [2], anaerobic endurance capacity [3], and skeletal muscle health [14-18]. The neuroprotective role of DHA as related to concussion and traumatic brain injury (TBI) risk has also been explored, with particular application to American football athletes [8-13].

As essential fats, EPA and DHA must be obtained exogenously because the human body has limited ability to synthesize these ω-3 FA from precursor ω-3 FA alpha-linolenic acid (ALA) [27]. Fish and seafood are the richest sources of ω-3 FA but there is wide variation in the EPA and DHA content of these foods, ranging from 100 mg to 2500 mg of combined EPA + DHA per three ounce serving [28-32]. Some of the richest food sources, such as salmon, may have up to ten times the EPA and DHA content compared with less rich sources like shrimp and tilapia [28-32]. Also of note, frequent consumption of some common sources of ω-3 FA may risk overexposure to mercury [28].

While there is currently no consensus for ω-3 FA dietary recommendations and athlete-specific recommendations have not been established [33-36], the Academy of Nutrition and Dietetics recommends consumption of 500 mg EPA + DHA daily, estimated to be accomplished via eating at least two servings of fish each week [33, 34]. To meet ALA needs, the National Academy of Medicine (formerly Institute of Medicine) recommends consumption of at least 1.6 g in adult men and 1.1 g daily in adult women [35]. Low ω-3 FA intake appears to be prevalent within the general population of North America [28, 29, 37, 38]. Reports of athletes’ ω-3 FA intake are minimal to date, but Wilson and Madrigal [39] observed daily intakes of EPA and DHA below 100 mg in a group of 58 National Collegiate Athletics Association (NCAA) Division I collegiate athletes. Little information is available at present with regard to athletes’ habitual use of ω-3 FA supplements.

In addition to ω-3 FA intake, ω-3 FA status may be evaluated using the Omega-3 Index (O3i), which reflects the sum of EPA and DHA in erythrocyte membranes as a percentage of total erythrocyte fatty acids [4]. Compared to other methods, O3i requires a minimum amount of blood (i.e., finger stick blood sample), has a low biological variability [40], is less affected by acute feedings to better reflect long-term ω-3 FA intake [41], and has been shown to correspond with ω-3 FA concentrations in the heart, brain, and a variety of other tissues [42, 43]. An O3i <4% has been associated with the highest risk for the development of cardiovascular disease; whereas, 4–8% is considered moderate risk and ≥8% is the lowest risk [4-6]. Recently, an average O3i of 4.4% was observed among collegiate football athletes at four U.S. universities [44]. A large scale assessment of O3i in collegiate athletes has not been described in the published literature, to our knowledge.

Prior to 2019, the NCAA classified ω-3 FA supplements as “impermissible”, which prevented athletic departments from purchasing such supplements for athletes unless prescribed by a team physician [45]. However, advocacy from member institutions facilitated a 2019 NCAA legislation change, reclassifying ω-3 FA supplements as permissible for athletic departments to purchase and provide for athletes [46]. As a result of this rule change, interest in and availability of ω-3 FA supplements has risen. In order to better inform recommendations and ultimately nutrition interventions, a better understanding of athletes’ ω-3 FA status is needed. Thus, the purpose of this study was to assess the ω-3 FA intake and O3i of male and female NCAA Division I athletes who participate in a variety of sports.

Methods

Study design

A multi-site, cross-sectional study was designed to assess the ω-3 FA dietary intake, ω-3 FA supplement use, and O3i of NCAA Division I athletes. These assessments were carried out during the 2018–2019 academic year.

Participants

In an effort to recruit a geographically diverse subject pool, the research team solicited volunteer research collaborators (registered dietitians or sports performance staff employed by their respective athletics programs) from the NCAA Division I Power 5 institutional membership (Atlantic Coast Conference, Big Ten Conference, Big Twelve Conference, Pacific-12 Conference, and Southeastern Conference). Research collaborators provided oversite for on-site data collection (in conjunction with the primary research team) and served as liaisons to the primary research team. One institution from each of eight regions throughout the U.S. was accepted (on a first come-first served basis), with the exception of the Northeast from which two were ultimately accepted since one became unable to complete blood measurements after agreeing to participate. The nine participating institutions represented the following states: California, Georgia, Illinois, Nebraska, Oregon, Pennsylvania, Texas, Utah and Virginia. Participating institutions were assured protection of program identity at the level of state.

Research collaborators posted flyers in common athlete facilities and sent email and/or text messages to all athletes within the institution, inviting them to participate in a research study aimed at assessing dietary and supplemental ω-3 FA intake. Male and female athletes who were over the age of 18 years and on a current roster for any NCAA Division I sport at one of the participating institutions were eligible to participate. Although there was some variation in sports offered at the participating institutions, all nine included men’s baseball, men’s and women’s basketball, men’s and women’s cross country, men’s football, men’s and women’s golf, women’s gymnastics, women’s soccer, women’s softball, men’s and women’s swimming & diving, men’s and women’s track & field, and women’s volleyball.

Omega-3 dietary assessment

A twenty six-item food frequency questionnaire (FFQ) validated to assess ω-3 FA dietary intake [39, 47] was administered to eligible participants electronically using Qualtrics (version XM, Provo, Utah, U.S.). Consent to participate was inferred by completion of the FFQ. The FFQ was modified to include demographic characteristics of participants (sex, age, academic year, and sport) and ω-3 FA supplement use. Within the FFQ, participants reported the frequency of consumption and average portion size for an extensive list of ω-3 FA food sources including fish, shellfish, walnuts, canola oil, flaxseed, flaxseed oil, and cod liver oil. For participants who indicated that they consumed ω-3 FA supplements, information about brand, form, dosage, and frequency taken was requested.

The FFQ results were compiled and analyzed using methodology outlined by Sublette et al [47]. Previously published databases [30-32] were used as a reference for ω-3 FA content of foods consumed based on source and portion size reported. Responses with more than one unanswered question were omitted from analyses.

Blood fatty acid analysis

Following completion of the dietary assessment portion of the study, participants were offered the opportunity to volunteer for a second portion of the study: analysis of blood fatty acids. A single drop of whole blood was collected from the index or middle finger and applied to a blood spot card pre-treated with an antioxidant cocktail. Blood collection was carried out by a registered dietitian or certified athletic trainer in an athletic training room or sports medicine facility. Samples were shipped to a central laboratory (OmegaQuant, Sioux Falls, South Dakota, U.S.) within fourteen days for analysis of fatty acids and calculation of the O3i using gas chromatography. This methodology was described in detail by Harris and Polreis [48].

Statistical analysis

Data were analyzed using IBM Statistical Package for the Social Sciences (SPSS) version 26. Descriptive statistics are expressed as means and standard deviations for continuous data, and frequencies and percentages for categorical data. Data were tested for normality using the Shapiro-Wilk test. Differences in outcomes between demographic groups were calculated using analysis of variance (ANOVA) or chi-square tests. Relationships between diet and blood variables were analyzed using Pearson’s correlations. Multiple regression analysis was used to assess the effects of diet on O3i with adjustment for demographic covariates including institution, sex, age, class year, and sport. Football vs non-football sport comparisons were made in order to compare results to existing literature, and because football programs may have different resources and athlete characteristics as compared with other sports. Significance was set at a level of p<0.05.

Ethical considerations

This study was approved by the Institutional Review Board of Virginia Tech (IRB# 18–606) and respective institutional research review committees. Consent for the dietary assessment portion of the study was inferred based on voluntary completion. Written and informed consent was provided by participants before starting the blood fatty acid portion of the study.

Results

In all, 1562 participants completed the dietary assessment portion of the study and 301 completed the blood analysis portion. Thirty-four FFQs were incomplete and thus, excluded from analysis. We also excluded 3 participants from the blood analysis since they did not have complete FFQ submissions. Overall, a total of 1528 participants (51% males) were included in dietary assessment analyses, and 298 (55% males) in the blood analysis portion. Descriptive characteristics of participants are shown in Table 1. Participants represented 15 different male sports and 19 different female sports from nine institutions. There were no differences in demographics between subject cohorts completing the dietary assessment and blood analysis portions of the study except that the blood cohort included 10 different male sports and 11 different female sports, and the Pennsylvania institution did not participate in the blood analysis (Table 1). These cohorts are representative of NCAA Division I athletes across the U.S. in respect to sex (NCAA Division I = 53% male, diet sample = 51% male, blood sample = 55% male) and mean age (NCAA Division I = 20.3 years, diet sample = 19.9 years, blood sample = 20.0 years). In order to protect anonymity and confidentiality of our high profile population, we have not made full data points publicly available.

Table 1

Descriptive characteristics of participants.

	Dietary Assessment	Blood Fatty Acid Analysis	Differences Test Statistic (p-value)
n	1,528	298
Sex (Male/Female)	Male: 780 (51.0%)	Male: 163(54.6%)	χ2 = 1.318/ (p = 0.251)
	Female: 748 (49.0%)	Female: 135 (45.3%)
Age (years; mean ± SD)	19.9 ± 1.4	20.0 ± 1.3	F = 1.610/ (p = 0.646)
Academic year n (%)	Freshman: 442 (28.9%)	Freshman: 88 (29.5%)	χ2 = 18.50/ (p = 0.470)
	Sophomore: 373 (24.4%)	Sophomore: 73 (24.4%)
	Junior: 377 (24.7%)	Junior: 70 (23.5%)
	Senior: 270 (17.7%)	Senior: 58 (19.4%)
	5th year or Graduate: 63 (4.1%)	5th year or Graduate: 9 (3.0%)
Sport n (%)	a Male Sports: 780 (51.0%)	c Male Sports: 163 (54.6%)	χ2 = 4.779/ (p = .1912)
	Football: 303 (19.8%)	Football: 81 (27.2%)
	Non-football: 477 (31.2%)	Non-football: 82 (27.5%)
	b Female Sports: 748 (49.0%)	d Female Sports: 115 (45.3%)
Region n (%)	California: 106 (6.9%)	California: 28 (28.6%)	χ2 = 7.003/ (p = .0991)
	Georgia: 158 (10.3%)	Georgia: 33 (11.1%)
	Illinois: 77 (5.0%)	Illinois: 29 (9.7%)
	Nebraska: 211 (13.8%)	Nebraska: 45 (15.1%)
	Oregon: 111 (7.3%)	Oregon: 39 (13.1%)
	Pennsylvania: 61 (4.0%)	Pennsylvania: 0 (0.0%)
	Texas: 336 (22.0%)	Texas: 40 (13.4%)
	Utah: 102 (6.7%)	Utah: 42 (14.1%)
	Virginia: 365 (23.9%)	Virginia: 43 (14.4%)

Baseball, Basketball, Cross Country, Fencing, Football, Golf, Gymnastics, Ice Hockey, Ski, Soccer, Swimming & Diving, Tennis, Track & Field, Volleyball, Wrestling

Basketball, Beach Volleyball, Bowling, Cross Country, Equestrian, Fencing, Field Hockey, Golf, Gymnastics, Lacrosse, Rifle, Rowing, Ski, Soccer, Softball, Swimming & Diving, Tennis, Track & Field, Volleyball

Baseball, Basketball, Cross Country, Football, Golf, Gymnastics, Tennis, Track & Field, Wrestling

Basketball, Cross Country, Fencing, Gymnastics, Lacrosse, Rifle, Rowing, Soccer, Softball, Swimming & Diving, Track & Field

Diet

Frequency of fish and seafood consumed and sources of fish and seafood consumed by participants are shown in Figs 1 and 2, respectively. A total of 601 participants (39%) met the recommendation to consume two or more fish servings weekly (Fig 1). Salmon and shrimp were the only EPA and DHA sources reported to be consumed by more than 50% of participants (Fig 2). Sources of ALA included canola oil (85%), walnuts (53.9%), chia (43.6%), flax or flax oil (34.9%), and cod liver oil (3.3%).

Fig 1

Frequency of fish and seafood consumption during the previous 6 months (n = 1528).

Fig 2

Sources of fish and seafood consumed during the previous 6 months (n = 1528).

Male participants consumed significantly more EPA and DHA than female participants and female participants consumed significantly more ALA than male participants (Table 2). Only 91 (6%) of participants consumed at least 500 mg combined EPA + DHA/day as advised by the Academy of Nutrition and Dietetics [33] and 62 (4%) met the National Academy of Medicine’s recommendation of 1.6 g ALA (men) and 1.1 g ALA (women) [35]. There were no significant differences found in EPA, DHA, ALA, EPA + DHA or total omega-3 intake when comparing individual sports with greater than 8 participants (men’s and women’s basketball, baseball, football, men’s and women’s soccer, men’s and women’s swim and dive, men’s track & field, wrestling and softball, women’s golf, women’s gymnastics, women’s lacrosse and women’s volleyball).

Table 2

Dietary consumption of Omega-3 fatty acids (n = 1528).

	Total Daily Intake (mg)	Sex		p-value
		Male n = 780	Female n = 748
EPA	46.8 +/- 86.9	53.4	40.4	.0042**
DHA	94.8 +/- 164.9	106.4	83.9	.0091**
ALA	571.8 +/- 1151.5	530.4	626.6	.0281*
EPA + DHA	141.7 +/- 250.6	159.8	124.3	.0068**

** p<0.01

Use of ω-3 FA supplements was reported by 229 participants (15%). A total of 153 (10%) purchased the supplement on their own, while 76 (5%) received supplements from their respective athletic program via physician prescription. Of the nine participating programs, seven were reported as providing supplements and of the 76 participants receiving supplements, 49 (69%) were football athletes. Most participants provided no response to brand, type, and dose of ω-3 FA supplements consumed.

Blood

Result of blood fatty acid and O3i analyses are shown in Table 3. O3i ranged from 2.25 to 7.23% (Fig 3), with 114 (38%) in the high risk category, 184 (62%) in the moderate risk category, and 0 (0%) in the low risk category (Fig 3). There were no significant differences in blood measures based on sex (Fig 4), institution, age, or academic year. There was no significant difference found in O3i when comparing individual sports with greater than 8 participants (men’s and women’s basketball, baseball, football, men’s and women’s soccer, women’s swim and dive, men’s track & field, wrestling and softball, women’s gymnastics, women’s lacrosse and women’s volleyball).

Table 3

Blood fatty acid analysis results (n = 298).

	Blood Fatty Acids (%)	Sex		p-value
		Male	Female
EPA	0.45 ± 0.19	53.4	40.4	p = 0.704
DHA	2.19 ± 0.59	106.4	83.9	p = 0.699
ALA	0.49 ± 0.19	530.4	626.6	p = 0.588
O3i	4.3 ± 0.81	4.3	4.4	p = 0.905

Fig 3

Distribution of Omega-3 index results (n = 298).

Ranges associated with risk for development of cardiovascular disease [4–6].

Fig 4

Omega-3 index in male and female NCAA division 1 student athletes.

Relationship between diet and blood measures

Dietary intake of both EPA and DHA were positively correlated with blood EPA, DHA, and O3i (Table 4). There was no correlation between dietary ALA intake and blood levels of EPA, DHA, ALA or O3i (Table 4).

Table 4

Diet and blood fatty acid correlations table.

	Diet EPA	Diet DHA	Diet EPA + DHA	Diet ALA	Diet Total ω-3	Blood EPA	Blood DHA	Blood ALA	Blood O3i
Diet EPA	1
Diet DHA	.977 **	1
Diet EPA + DHA	0.990 **	0.997 **	1
Diet ALA	0.134	0.154	0.148	1
Diet Total ω-3	0.332 *	0.552 *	0.347 *	0.979 **	1
Blood EPA	0.342 *	0.334 *	0.338 *	0.296	0.339 *	1
Blood DHA	0.397 *	0.404 *	0.403 *	0.214	.273	.402 *	1
Blood ALA	0.072	0.080	0.078	0.090	0.098	0.072	-0.122	1
Blood O3i	0.437 *	0.441 *	0.442 *	0.271	0.332 *	0.648 **	0.958 **	-0.079	1

*p<0.05,

**p<0.01

After controlling for institution, sex, age, class year and sport (football vs. non-football), frequency of fish and seafood consumption was a significant predictor of O3i (R2 = .3701, p<0.01). Each additional serving of seafood was associated with a O3i increase of 0.27% (Fig 5). Participants who reported taking ω-3 FA supplements had significantly higher O3i compared with those not taking supplements (4.7 vs. 3.7%, respectively; p<0.05). Participants who met the Academy of Nutrition and Dietetics’ recommendation of 500 mg EPA + DHA per day had a higher O3i compared to those who consumed less than the daily 500 mg EPA + DHA recommendation (5.4% vs. 4.3%, p<0.05).

Fig 5

Association between fish or seafood meal frequency and omege-3 index (n = 298).

After controlling for institution, sex, age, class year and sport (football vs. non-football), frequency of fish and seafood consumption was a significant predictor of O3i (R2 = .3701, p<0.01). Each additional serving of seafood was associated with a O3i increase of 0.27%.

Discussion

The primary goal of this study was to describe the ω-3 FA status of NCAA Division I athletes in the U.S. Our findings indicate that most NCAA Division I athletes do not meet current dietary recommendations for ω-3 FA and have sub-optimal O3i as compared to currently proposed cardiovascular benchmarks. To our knowledge, this is the first large scale assessment of ω-3 FA status of male and female collegiate athletes from a variety of sports.

Given the pattern of inadequate ω-3 FA status observed among NCAA Division I athletes, clinicians should consider nutritional interventions aimed at improving ω-3 FA status. One strategy could be increasing consumption of fish and seafood, the richest sources of EPA and DHA, as nearly half of participants reported no fish consumption in the last 6 months. Based on our findings, more frequent inclusion of ω-3 FA-rich sources in provided meals is an encouraged method for improving these low intakes. Capitalizing on popular fish and seafood sources (salmon, shrimp, crab, tuna, and tilapia were consumed the most in the current study) may be beneficial [49]. However, those involved in nutrition programming and meal planning should consider the independent omega-3 profiles of these foods. Emphasizing the richest sources of DHA and EPA by incorporating sources like salmon, trout and mackerel, for example, provide a more concentrated dose aimed at improving O3i status. Practitioners should also recognize that plant-based sources of ω-3 FA are only rich in ALA and that the conversion of ALA to EPA and DHA is minimal [27]. The observed lack of correlation between dietary ALA and blood measures of EPA, DHA and O3i in the current study, is also consistent with previous findings [39, 47].

In recent years, the NCAA has seen significant changes in terms of the feeding opportunities available for Division I athletes as a result of the deregulation of meal restrictions in 2014, allowing institutions more flexibility in the provision of nutrition to athletes [50]. It is important to note that this legislation change was specific to Division I athletes, and Division II and III (in addition to non-Power Division I) athletes may face greater obstacles in terms of meeting adequate intake as a result of each program’s unique financial limitations.

Although the majority of collegiate athletes participating in the present study did not meet current dietary ω-3 FA recommendations—similar to previous observations [39]—these guidelines are not specific to athletes. Further research is needed to establish athlete-specific recommendations, taking into consideration the physiological implications of advanced levels of training on metabolism and the inflammatory response [51-53]. For example, lower average O3i was observed among non-elite runners with greater training mileage compared to those with lesser running mileage [51]. Additional research is also needed to identify intake of ω-3 FA most effective for neuroprotection and brain health.

No participant in the current study, including those who consumed fish or seafood twice or more per week, had an O3i of 8%, the level associated with lowest cardiovascular disease risk [4-6]. Thus, achieving optimal ω-3 FA status through diet alone may be difficult and it is plausible that athletes may actually have higher needs than the general population. The use of ω-3 FA supplements is another strategy for improving ω-3 FA status, and has been discussed as a potentially helpful nutritional tool for athletes [54]. A small percentage of participants reported ω-3 FA supplement use but almost none were able to provide information about brand, form, dosage, and frequency of supplements used. The recent NCAA guidelines changes [55] present an opportunity to more readily provide ω-3 FA when appropriate, and to do so in a safe, controlled, and monitored fashion.

The sub-optimal O3i observed for in our study (4.3%) was similar to previous observations [39, 44, 56, 57], and did not differ based on sex or sport. Football-specific findings in this study were virtually identical to previous findings in Division I football athletes with both groups averaging an O3i of 4.4% [44]. While further research is needed to investigate potential differences in needs between athletes of different sex and sport, we observed NCAA Division I athletes collectively have low ω-3 FA status. Interestingly, the higher consumption of EPA and DHA observed in male participants compared to females did not translate to higher O3i values. This might suggest external factors such as higher average body mass, higher caloric needs and availability of athletic department nutrition resources drove the observed increases in EPA and DHA intake and was not significant enough to impact blood status. To our knowledge, no U.S.-based athletes have been documented in the peer reviewed literature as having O3i greater than 8% [39, 44], the proposed benchmark for optimal cardiovascular health [4-6]. Research suggests EPA and DHA may reduce cardiovascular risk factors such as dyslipidemia and high blood pressure[58-60] and even sudden death related to cardiac causes [61-65], which are applicable to a wide variety of athletes. This will continue to be of particular interest for higher mass athletes (such as linemen in American football) observed to be at higher risk of cardiovascular disease and metabolic syndrome [61, 66, 67]. Given this concern, in combination with the associated health functions of ω-3 FA related to both athlete performance and well-being [2, 3, 8–13, 15–24, 26], a focus on improved O3i is warranted. It is important to note, however, that target O3i for non-cardiovascular conditions is not well-established and continuing research is needed to investigate the impact of O3i on athlete health and performance measures.

Strengths & limitations

Collaboration with a diverse group of Power 5 institutions enabled us to study a large sample of athletes from nearly every NCAA sport with varying dietary habits and available resources. Further, given the timing of the NCAA legislation changes in relation to the timeline of our assessment, this investigation serves as a baseline for ω-3 FA intake and ω-3 FA supplement use among NCAA Division I athletes. Finally, our results parallel those of others who have observed a positive correlation between dietary EPA and DHA intake and O3i [68-70]. This suggests that the FFQ used was a reliable measure of ω-3 FA intake [39, 47]. This FFQ provides a cost-effective method for assessing ω-3 FA status in clinical situations where blood assessment may not be practically or financially warranted.

The study does have some limitations, however. In an effort to recruit a large, geographically-diverse cohort and provide equitable experiences for all participants, we included athletes from all sports sponsored by each institution in the study. Although we achieved a large and very diverse cohort (with male/female representation similar to that of the overall NCAA Division 1 membership), there was variability in number of participants from each sport and it was difficult to make sport by sport comparisons. With this in mind, we categorized sports as football vs. non-football for many analyses in order to compare our data with other published results [44]. As there are characteristics that often distinguish football from other intercollegiate sports, such as programmatic resources, athlete size, and occurrence of head injury, we believe this categorization is relevant. It should be acknowledged that while participating institutions were assured anonymity, inclusion of state and Power 5 criteria does allow the reader to make some assumptions about the identity of institutions. Although we believe that our results are generalizable to NCAA Division I athletes, further sports-specific investigations would be valuable, as would evaluation of Division II and III athletes.

Regarding the dietary assessment, fish and seafood vary in nutritional content based on a number of factors, including variety consumed, location, and time of year. Our assessment did not account for this variation. Additionally, we did not collect data related to race/ethnicity, height, and body weight in effort to assure anonymity of participants, but this information may have been insightful in data analysis. Overall, the lack of universally accepted dietary recommendations and blood measure standards provided an additional obstacle in terms of interpreting our results, which should be a primary motive for future research.

Conclusion

Prior to the 2019 change in NCAA legislation change related to ω-3 FA supplementation, we observed sub-optimal omega-3 status in NCAA Division I athletes based on both dietary and blood assessments. These results serve to inform future nutritional interventions aimed at improving ω-3 FA status among athletes. Results also provide a baseline in order to measure the impact of nutrition interventions created as a result of this legislation change.

12 Feb 2020

PONE-D-20-02046

Dietary and Biological Assessment of Omega-3 Status of Collegiate Athletes: A Cross-Sectional Analysis

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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

**********

5. 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: PONE-D-20-02046 presents results from a sample of collegiate athletes. While some parts of this manuscript were interesting, other areas could be improved. I hope the authors consider my feedback for enhancing their manuscript.

MAJOR COMMENTS

• Introduction: It is generally rare to insert tables such as these in Introduction sections (probably more so relevant for thesis and dissertation papers). A lot of this information could be abbreviated and inserted where appropriate in the text. While I understand the information the authors are trying to convey here, it just comes across as odd and distracting to this reviewer.

• Participants: How were the institutions selected within each state, and why were these specific states selected (e.g., Illinois vs. Minnesota)? Can specific institutions also be identified?

• Methods: More information about sampling would be helpful. For example, was a snowball sampling technique used? How were missing data on questionnaires treated? How were questionnaire administered (e.g., in-person)?

• Statistical analysis: More detail about the statistical analyses and how it is compatible with what is presented in the results is needed. For example, what specific demographic covariates were in the multiple regression analysis?

• Results: Why were data dichotomized by football for most tables? By doing this, you are also merging sexes in the non-football category. This could be problematic because there appears to be significant differences between sexes (e.g., Table 4).

MINOR COMMENTS

• Line 5: Be sure to use consistency with in-text citations in the manuscript “,(1-6)” vs “(14-18),”.

• Table 1: Consider instead listing frequency and percent for sex, similar to what was done for academic year, etc.

• Methods: Can you confirm in the manuscript that each participating institution had the same sports offered across institutions?

• Figures 4 and 5: Please insert measures of variability in the figure.

• Figure 6 may need a new title. “Relationship” suggests there was a statistical test performed. The figure does not represent this in its current form.

• Discussion: Could the authors speculate how these findings would generalize to non-power 5 athletes and athletes in Divisions II and III?

Reviewer #2: The objective of this study was to characterize dietary intake of omega-3’s and determine the omega-3 index, a measure of EPA and DHA in red blood cell membranes, in collegiate athletes. Similar to the general population, very few athletes met recommendations for diet or the omega-3 index. These findings are interesting as the NCAA recently (2019) began permitting the use of omega-3 supplements. Discussion on why the NCAA reclassified omega-3’s would be helpful. Additionally, the authors discuss their findings in the context of cardiovascular disease, but this should be justified, as I suspect collegiate athletes are a lower risk of cardiovascular disease compared to the general population. Additional suggestions are included below:

• Was there a rationale for the football, non-football male sport, and female sport grouping? It may be worth mentioning the relationship between traumatic brain injury and omega-3 supplementation.

• The authors discuss the omega-3 index in terms of cardiovascular disease, which I assume is low in this population (collegiate athletes) compared to the general population. Please justify.

• Line 17: Remove table 1, provide a reference (USDA nutrient database), and consider listing the range of EPA/DHA in the context of commonly consumed choices.

• Line 33: Remove table 2 and include key references in the text body.

• Line 87: Were all participants invited to participate in the second phase of the study? If not, were the invitations to participate randomized?

• Line 129: Consider, “601 participants (39%) consumed at least two or more servings of fish weekly.”

• Line 137: If available, it may be worth noting which teams provided supplements vs. which teams did not.

• Line 165: Should this be figure 6, not figure 5?

• Line 218: reference (55) is cardiovascular risk of American football athletes not all. The review article (55) that is cited says “This review was constructed to delineate our contemporary understanding of cardiovascular health among American-Style Football participants”.

• Figures 4-6 should include error bars and statistical analyses should be conducted.

**********

6. 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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Reviewer #1: No

Reviewer #2: 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 to be viewed.]

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10 Mar 2020

Additional Requirements

Feedback Author Response

1 Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We have reviewed the manuscript in detail to meet PLOS ONE’s style requirements. Multiple revisions were made, particularly in the reference section.

2 In your Methods section, please provide additional information on how the participating institutions were selected.

Moreover, please ensure you have provided sufficient details to replicate the analyses such as: a) a statement as to whether your sample can be considered representative of a larger population, b) a description of how participants were recruited, and c) descriptions of where participants were recruited and where the research took place.

Thank you for the opportunity to clarify the methodology used for the study. Several edits have been made within the Methods section, including the requested details.

Of note, our study cohort represents almost 1% (1528/≈160,000) of the NCAA Division 1 student-athlete population and 5% (1528/≈30,000)of the NCAA Division 1 Power 5 student-athlete population. Institutions from throughout the U.S. were included among our study cohort (intentionally representing the 8 geographic regions), our gender breakdown matches that of the NCAA Division 1 student-athlete population, and almost every NCAA Division 1 sport was included among our cohort. Thus, we feel that our cohort is a strong representation of the population. However, we readily acknowledge that different sampling methodology may have led to a more perfectly representative cohort. We have added this as a limitation of the study.

3 Thank you for stating in your Acknowledgements Section:

"This study was funded, in part, by the Collegiate and Professional Sports Dietitians Association Research Award."

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

We have modified the funding statement to indicate that the only funding received for the study was from the CPSDA Research Award, and that no additional external funding was received. The “in part” phrase is incorrect!

4 Please ensure that you refer to Figure 6 in your text as, if accepted, production will need this reference to link the reader to the figure. Thank you for your catching this typo. This figure is now properly labeled within the text.

5 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.

While we appreciate and support PLOS ONE’s commitment to data transparency, we have not made full data tables available on a public repository. This is because we assured participating institutions anonymity for themselves and their student-athletes who are considered a high profile, sensitive population. A statement to this effect was added to the results section in the manuscript.

We are willing to discuss this issue further with the editor, reviewers, and even the IRB and participating institutions if necessary.

Reviewer 1

Feedback Author Response

MAJOR COMMENT 1 Introduction: It is generally rare to insert tables such as these in Introduction sections (probably more so relevant for thesis and dissertation papers). A lot of this information could be abbreviated and inserted where appropriate in the text. While I understand the information the authors are trying to convey here, it just comes across as odd and distracting to this reviewer. We have removed tables from the introduction and incorporated key abbreviated information int the text. This edit does improve the flow and readability of the introduction – thank you.

MAJOR COMMENT 2 Participants: How were the institutions selected within each state, and why were these specific states selected (e.g., Illinois vs. Minnesota)? Can specific institutions also be identified? We have clarified institution recruitment within the text; thank you for this recommendation. Briefly, we solicited volunteer institutions from within the NCAA Division I Power 5 membership. On a first come/first served basis, we accepted one institution from each of eight pre-defined regions within the U.S. (Northwest, Southwest, etc.). We ultimately accepted two Northeast programs since the first became unable to collect blood samples due to a staffing change.

Prior to the start of the study, participating institutions were promised protection of program identity at the level of state. Thus, programs were identified only by state (and NCAA Division I Power 5).

MAJOR COMMENT 3 Methods: More information about sampling would be helpful. For example, was a snowball sampling technique used? How were missing data on questionnaires treated? How were questionnaire administered (e.g., in-person)? Additional details have been added throughout the methods section. For example, we have noted that student-athletes volunteered to participate through convenience sampling, which included open recruitment of all student-athletes at each institution (flyers in common/shared athletics facilities, email, and text messages). Questionnaires were administered electronically and responses with more than one question left blank were excluded from analyses (n=34).

MAJOR COMMENT 4 Statistical analysis: More detail about the statistical analyses and how it is compatible with what is presented in the results is needed. For example, what specific demographic covariates were in the multiple regression analysis? We have clarified language around the statistical analyses we used. Demographic covariates (institution, sex, age, and class year, and sport) were added to the text.

MAJOR COMMENT 5 Reviewer 1) Why were data dichotomized by football for most tables? By doing this, you are also merging sexes in the non-football category. This could be problematic because there appears to be significant differences between sexes (e.g., Table 4).

The research team revisited this important issue and made several modifications. Realizing that our study design was likely to yield a large number of total athletes (but not necessarily a large or equivalent number of student-athletes from each different sport), our study plan included analysis of football data vs. other sport data. This would allow for comparison of our results to the existing literature, which primarily involves football (ex: Anzalone et al., 2019). Furthermore, other characteristics commonly distinguish football from other intercollegiate sports, including programmatic resources (budget, meals, supplements, staff, etc.), athlete size, and occurrence of head injury (though we acknowledge that head injury is an issue for many athletes from many different sports).

However, upon re-examining the results, we decided to keep some football vs. non-football comparisons, but to minimize these throughout the manuscript (removing it from the tables). Although the significant difference in football vs. non-football athletes remained in EPA + DHA when controlling for sex (regression analysis), we realize that the comparison may not be ideal since data on resource differences, athlete size, etc. were not collected.

If the reviewer would like to see additional changes made to our analysis and interpretation, we welcome the option to consider.

MINOR COMMENT 1 Be sure to use consistency with in-text citations in the manuscript “,(1-6)” vs “(14-18),”. Citation formatting has been edited to consistently reflect PLOS ONE’s formatting requirements. We apologize for neglecting this important detail in the initially submitted manuscript.

MINOR COMMENT 2 Table 1: Consider instead listing frequency and percent for sex, similar to what was done for academic year, etc. This edit has been made in Table 1.

MINOR COMMENT 3 Methods: Can you confirm in the manuscript that each participating institution had the same sports offered across institutions? Unfortunately, each participating institution did not offer all of the same sports, but there was a great deal of overlap. All nine institutions offer M baseball, M & W basketball, M & W cross country, M football, M & W golf, W gymnastics, W soccer, W softball, M & W swimming & diving, M & W track & field, and W volleyball. We considered including only student-athletes from these sports. However, since our hypothesis was that there would be no difference in ω-3 FA status based on sport, we elected to invite student-athletes from all teams to participate in this cross sectional study (which helped to facilitate a large sample size). Our results indicate that differences in ω-3 FA status seem to be based on gender rather than sport. Additionally, research collaborators and the research team felt that inviting all athletes on all teams to participate was presented the best equitability to student-athletes, which is a priority among NCAA institutions.

We have added language to the Methods section indicating that there were some differences in sports offered at each institution and also added this issue as a study limitation.

MINOR COMMENT 4 Figures 4 and 5: Please insert measures of variability in the figure.

Measures of variability and error bars have been added. Thank you for this important recommendation.

MINOR COMMENT 5 Figure 6 may need a new title. “Relationship” suggests there was a statistical test performed. The figure does not represent this in its current form.

We have edited the title of the figure to refer to Association rather than Relationship. We also added a footnote to refer to the results of the regression analysis conducted on the fish/seafood frequency and O3i data.

MINOR COMMENT 6 Discussion: Could the authors speculate how these findings would generalize to non-power 5 athletes and athletes in Divisions II and III? This is an important recommendation, which we appreciate. Information about generalizability of our results was added to the discussion.

Reviewer 2

Feedback Author Response

GENERAL REMARKS Discussion on why the NCAA reclassified omega-3’s would be helpful.

A statement was added to the introduction related to this remark. Member institution advocacy (motivated by health and safety benefits of ω-3 FA) led to the NCAA reclassification.

COMMENT 1 Was there a rationale for the football, non-football male sport, and female sport grouping? It may be worth mentioning the relationship between traumatic brain injury and omega-3 supplementation.

Thank you for the important question and suggestion. We have added mention of the relationship between brain health and ω-3 FA.

Additionally, the research team revisited the football vs. non-football classifications and made several modifications. Realizing that our study design was likely to yield a large number of total athletes (but not necessarily a large or equivalent number of student-athletes from each different sport), our study plan included analysis of football data vs. other sport data. This would allow for comparison of our results to the existing literature, which primarily involves football (ex: Anzalone et al., 2019). Furthermore, other characteristics commonly distinguish football from other intercollegiate sports, including programmatic resources (budget, meals, supplements, staff, etc.), athlete body size, and occurrence of head injury (though we acknowledge that head injury is an issue for many athletes from many different sports).

However, upon re-examining the results, we decided to keep some football vs. non-football comparisons, but to minimize these throughout the manuscript (including removing it from the tables). Although the significant difference in football vs. non-football athletes remained in EPA + DHA when controlling for sex (regression analysis), we realize that the comparison may not be ideal since data on resource differences, athlete size, etc. were not collected.

If the reviewer would like to see additional changes made to our analysis and interpretation, we welcome the option to consider.

COMMENT 2 The authors discuss the omega-3 index in terms of cardiovascular disease, which I assume is low in this population (collegiate athletes) compared to the general population. Please justify.

Although O3i reflects the ω-3 FA content of a variety of tissues, a strong association between, O3i is only strongly associated with cardiovascular disease risk in the literature.

The risk of cardiovascular disease and metabolic syndrome is elevated in some college athletes (such as football) and the risk of sudden cardiac death is elevated in many sports, which is why our O3i results are especially important.

Modifications have been made to the manuscript to reflect these points.

COMMENTS 3 & 4 Introduction: Remove table 1, provide a reference (USDA nutrient database), and consider listing the range of EPA/DHA in the context of commonly consumed choices.

Introduction: Remove table 2 and include key references in the text body. We have made the recommended edits. Removing the tables and adding a summary/references has made for a much more readable introduction. Thank you!

COMMENT 5 Methods: Were all participants invited to participate in the second phase of the study? If not, were the invitations to participate randomized? The methods section has been edited to add more details, including information about recruitment. All participants who completed the dietary assessment were invited to participate in the blood analysis portion of the study. The exception to this is the Pennsylvania institution for whom blood analysis was not conducted due to staffing changes.

COMMENT 6 Results: Consider, “601 participants (39%) consumed at least two or more servings of fish weekly.”

This edit has been made.

COMMENT 7 Results: If available, it may be worth noting which teams provided supplements vs. which teams did not This data is interesting, and we have added it to the result section. Of the supplements provided by athletic programs, 7 of 9 did provide supplements and 49 (69%) were to football athletes.

Because of the existing NCAA legislation at the time of data collection, no institutions were able to provide ω-3 FA supplementation without a physician prescription.

COMMENT 8 Line 165: Should this be figure 6, not figure 5? Thank you for catching this typo. The statement has been corrected.

COMMENT 9 Line 218: reference (55) is cardiovascular risk of American football athletes not all. The review article (55) that is cited says “This review was constructed to delineate our contemporary understanding of cardiovascular health among American-Style Football participants”. We modified this statement to indicate football-specific observations, and also added additional references.

COMMENT 10 Figures 4-6 should include error bars and statistical analyses should be conducted. These have been added where appropriate. Thank you.

Submitted filename: PLOS ONE response to reviewers 3.8.2020.docx

Click here for additional data file.

2 Apr 2020

PONE-D-20-02046R1

Dietary and Biological Assessment of Omega-3 Status of Collegiate Athletes: A Cross-Sectional Analysis

PLOS ONE

Dear Dr. Rockwell,

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 points raised during the review process.

Please address the following:

List each Power 5 Conference for clarity to readers

Expand upon limitations

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Emily Sauers

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

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 #1: (No Response)

Reviewer #2: Yes

**********

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

Reviewer #1: (No Response)

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 #1: (No Response)

Reviewer #2: No

**********

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: (No Response)

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 #1: • Please specifically list each Power 5 conference in the area in which it is first introduced to provide specificity to readers that may not know these conferences in a paratheatrical note.

• The authors indicate that participating institutions were assured protection of program identity; however, it is known that some states that only have one Power 5 institution will be identified by default (e.g., University of Utah). This should be listed as a limitation.

• The authors did a nice job of addressing the concern related to comparing football and non-football athletes in their revisions letter; however, the limitations of making these comparisons should also be listed in the limitations paragraph.

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 #1: No

Reviewer #2: 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 to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Apr 2020

Reviewer #1:

• Please specifically list each Power 5 conference in the area in which it is first introduced to provide specificity to readers that may not know these conferences in a paratheatrical note. This edit has been made.

• The authors indicate that participating institutions were assured protection of program identity; however, it is known that some states that only have one Power 5 institution will be identified by default (e.g., University of Utah). This should be listed as a limitation. This edit has been made.

• The authors did a nice job of addressing the concern related to comparing football and non-football athletes in their revisions letter; however, the limitations of making these comparisons should also be listed in the limitations paragraph. Thank you. This edit has also been made.

Submitted filename: Response to Reviewers 4.3.2020.docx

Click here for additional data file.

8 Apr 2020

Dietary and Biological Assessment of the Omega-3 Status of Collegiate Athletes: A Cross-Sectional Analysis

PONE-D-20-02046R2

Dear Dr. Rockwell,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Emily Sauers

Academic Editor

PLOS ONE

17 Apr 2020

PONE-D-20-02046R2

Dietary and Biological Assessment of the Omega-3 Status of Collegiate Athletes: A Cross-Sectional Analysis

Dear Dr. Rockwell:

I am 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.

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With kind regards,

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on behalf of

Dr. Emily Sauers

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PLOS ONE