Dietary Supplements for Athletic Performance in Women: Beta-Alanine, Caffeine, and Nitrate

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Molly J. Murphy Department of Nutrition, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

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Blake R. Rushing Department of Nutrition, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Nutrition Research Institute, The University of North Carolina at Chapel Hill, Kannapolis, NC, USA

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Susan J. Sumner Department of Nutrition, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Nutrition Research Institute, The University of North Carolina at Chapel Hill, Kannapolis, NC, USA

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Anthony C. Hackney Department of Nutrition, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Department of Exercise & Sport Science, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

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Beta-alanine, caffeine, and nitrate are dietary supplements generally recognized by the sport and exercise science community as evidence-based ergogenic performance aids. Evidence supporting the efficacy of these supplements, however, is greatly skewed due to research being conducted primarily in men. The physiological differences between men and women, most notably in sex hormones and menstrual cycle fluctuations, make generalizing male data to the female athlete inappropriate, and potentially harmful to women. This narrative review outlines the studies conducted in women regarding the efficacy of beta-alanine, caffeine, and nitrate supplementation for performance enhancement. Only nine studies on beta-alanine, 15 on caffeine, and 10 on nitrate in healthy women under the age of 40 years conducted in normoxia conditions were identified as relevant to this research question. Evidence suggests that beta-alanine may lower the rate of perceived exertion and extend training bouts in women, leading to greater functional adaptations. Studies of caffeine in women suggest the physiological responder status and caffeine habituation may contribute to caffeine’s efficacy, with a potential plateau in the dose–response relationship of performance enhancement. Nitrate appears to vary in influence based on activity type and primary muscle group examined. However, the results summarized in the limited literature for each of these three supplements provide no consensus on dosage, timing, or efficacy for women. Furthermore, the literature lacks considerations for hormonal status and its role in metabolism. This gap in sex-based knowledge necessitates further research on these ergogenic supplements in women with greater considerations for the effects of hormonal status.

Dietary supplements marketed as “ergogenic aids” are increasingly popular among international and U.S. collegiate and elite-level athletes. Ergogenic dietary supplements are promoted with claims of enhancing performance by improving strength, endurance, or other physiological attributes, though the range of supplements on the market far exceeds the number whose efficacy have been adequately studied and reported (Maughan et al., 2018; Molinero & Márquez, 2009). It is estimated that the rate of supplement consumption in athletes is higher than that of the general U.S. population, with the highest use in elite athletes compared with nonelite athletes (Knapik et al., 2016). Studies of college athletes suggest that between 64% and 89% regularly use at least one dietary supplement (Froiland et al., 2004; Knapik et al., 2016; Osterman et al., 2020).

Most dietary supplement use appears to be similar between men and women, with a few exceptions (Knapik et al., 2016). However, the current body of evidence supporting many of the most commonly used supplements has primarily investigated the effect of these ergogenic aids on performance in men. Despite the growing understanding that women are not small men, female and female subgroup studies continue to be outnumbered by male only or mixed sex studies in sports performance and nutrition literature (Hackney, 2017). Sex-specific differences, specifically hormonal changes during the female menstrual cycle, may have implications in the efficacy of oral nutritional supplements intended for performance enhancement. Five ergogenic supplements are identified by the American College of Sports Medicine and the International Olympic Committee as having evidence-based uses in sport nutrition; caffeine, creatine, beta-alanine, sodium bicarbonate, and nitrate (Maughan et al., 2018; Thomas et al., 2016a,b). Of these, beta-alanine, caffeine, and nitrate present a strong disparity in sex-specific research and thus have been chosen as the focus of this review. While Wickham and Spriet (2019) were the first to summarize the potential sex-based differences in nitrate supplementation, to date, no such reviews exist for caffeine or beta-alanine. Physiologically, these ergogenic aids work on different bodily systems and involve varying mechanisms for effect, and as such are utilized for training and competition in various athletic activities. Though each supplement functions differently and is investigated as an individual, rather than concurrent, means to improve performance, including all three in this review aims to draw attention to the multifaceted gap in female-based dietary and exercise literature.

A study on substance use in National Collegiate Athletic Association (NCAA) athletes reported that 12.1% of collegiate athletes use supplemental amino acids (AA) for ergogenic purposes (NCAA, n.d.). Beta-alanine is one such amino acid that has been studied for its performance-enhancing properties. Beta-alanine is a nonessential, nonproteinogenic AA. Rather, it is the rate-limiting precursor to the dipeptide carnosine, which is stored in skeletal muscle for use as a pH buffer. During high-intensity exercise, lactate production causes accumulation of hydrogen (H+) ions, lowering the pH of the skeletal muscle and leading to muscular fatigue (Hobson et al., 2012). Carnosine acts as an H+ buffer to maintain the pH of muscle fibers and prevent fatigue-inducing acidification (Hobson et al., 2012). Elevating intramuscular carnosine levels, therefore, should increase the muscle’s buffering capacity, which delays the onset of fatigue and allows for sustained high-intensity exercise. As the rate-limiting step in carnosine synthesis, increasing the availability of beta-alanine has been found to increase carnosine levels (Trexler et al., 2015). The practical effects of this increase in carnosine on training and performance are unconfirmed and remain an area of study. Internationally, consensus statements on the efficacy of beta-alanine supplementation vary; however, the position statements of The Academy of Nutrition and Dietetics, Dietitians of Canada, and the American College of Sports Medicine, and the International Society of Sports Nutrition support the AA’s role in enhancing exercise performance (Thomas et al., 2016b; Trexler et al., 2015). Notably, a 2017 systematic review and meta-analysis by Saunders et al. (2017) outlines beta-alanine’s efficacy in primarily male studies.

Caffeine is one of the most used ergogenic aids in sport, with 28.6% of NCAA athletes reporting use of caffeinated energy drinks (NCAA, n.d.). Caffeine is a plant-derived central nervous system stimulant. It is easily and rapidly absorbed in the body, and, as a lipid-soluble compound, crosses the blood–brain barrier (Goldstein, Ziegenfuss et al., 2010). Caffeine functions as an adenosine receptor antagonist, increasing release of stimulating neurotransmitters, such as acetylcholine and norepinephrine and leading to enhanced arousal and reduced fatigue. Caffeine is also purported to function as an ergogenic aid by promoting fatty acid mobilization and shifting substrate utilization away from glycogen (Ganio et al., 2009; Goldstein, Ziegenfuss, et al., 2010). Because caffeine is distributed throughout both the central and peripheral systems, there are several other potential mechanisms of action by which caffeine may enhance performance. Unlike with beta-alanine, international consensus offers substantial support for the efficacy of caffeine as an ergogenic aid, especially in aerobic forms of exercise (Goldstein, Ziegenfuss, et al., 2010; Guest et al., 2021; Thomas et al., 2016b).

Dietary nitrate is emerging as an ergogenic supplement with potential to improve performance via increased oxygen delivery. Beetroot and beetroot juice are the primary supplemental forms of inorganic nitrate (NO3). Upon consumption, the nitrate is first converted to nitrite (NO2) and subsequently reduced to nitric oxide (NO) (Lundberg et al., 2008). One of the multiple biological functions of NO, a free radical, is to stimulate vasodilation of blood vessels, improving oxygen delivery to muscles. Dietary nitrate supplementation has also been shown to improve oxygen consumption during exercise, possibly by limiting the uncoupling actions of the oxidative phosphorylation system and diverting membrane potential toward adenosine triphosphate (ATP) synthesis to improve mitochondrial efficiency (Larsen et al., 2011; Lidder & Webb, 2013). Nitrate has been shown to enhance both sustained submaximal aerobic exercise and short-duration high-intensity exercise, though more data are needed to clarify optimal dosage and timing of supplementation (Hoon et al., 2013; Maughan et al., 2018). Macuh and Knap (2021) reviews nitrate’s effects on exercise performance in studies conducted in males or without stratification by sex.

It is important to expand on current male-centric evidence by way of female-specific research on dietary supplements and performance measures as it is well established that differences in female sex hormones can affect signaling and metabolic pathways related to exercise. Resting metabolic rate has been found to be elevated in the high-estrogen and high-progesterone luteal phase (Benton et al., 2020). Phase differences in estrogen and progesterone also impact substrate utilization, as outlined in a 2010 review by Oosthuyse and Bosch. Evidence suggests that estrogen promotes catecholamine sensitivity, leading to increased rate of lipolysis, as well as stimulates adenosine monophosphate-kinase (AMP-kinase) activity to promote free fatty acid uptake. Protein catabolism fluctuates across menstrual phases, with AA oxidation higher in luteal phase compared with follicular phase. This latter review posits that progesterone may have the greatest impact on increased AA catabolism, while estrogen may reduce AA oxidation. Estrogen is also understood to impact glucose uptake; estrogen both promotes insulin sensitivity and contraction-stimulated glucose uptake into Type 1 muscle fibers. Progesterone antagonizes this uptake and promotes insulin resistance. Thus, the relative increase in estrogen compared with progesterone based on phasic hormone levels determines the degree of influence that the hormones have on glucose metabolism (Hackney, 2021; Oosthuyse & Bosch, 2010). In light of these known variations in metabolism based on changes in female sex hormones, studies controlling for or comparing menstrual phases in women are important to understand how ergogenic aids and exercise performance may be impacted differently at different moments in a women’s menstrual cycle given that training and competition may occur at any point in this hormonal cycle. While male sex hormones remain relatively stable, the significant fluctuations in sex hormones in women necessitate women-only studies to formulate dietary supplement guidelines for the female athlete (Hackney & Viru, 2008; Moore et al., 2021).

Beta-alanine, caffeine, and nitrate are prime examples of highly prevalent ergogenic aids that are advertised and used without strong evidence of efficacy in women. Sport and nutrition professional associations have published guidelines for supplementation that, though sometimes based on bodyweight, do not differentiate between men and women, implying that no sex-based differences exist though this has not been proven (Maughan et al., 2018). In light of these guidelines, and in accordance with the current understanding of metabolic variations across the menstrual cycle, the purpose of this review is to provide a summary of studies conducted to investigate the efficacy of beta-alanine, caffeine, and nitrate as ergogenic aids in female athletes, both recreational and elite. By summarizing the current literature, this review aims to highlight the disparity in ergogenic research in females and present considerations for further sex-specific research trials.

Methods

Relevant publications for this review were found using three search strings across four databases. The search string “(female OR woman OR women) AND (athlete OR sport) AND (beta alanine OR β-Alanine) AND (supplement OR supplementation)” was entered into the PubMed, SPORTDiscus, Scopus, and Google Scholar databases. For each supplement, the same search string was used, with the addition of the supplement-specific keywords: for caffeine, the search included only “caffeine,” and for nitrate the search included “nitrate OR beetroot.” The search was filtered for randomized control trials from the Year 2000 to March 2021 to emphasize the most up-to-date literature available. In keeping with the focus of this review, articles that included participants with a mean age >40 years or those with preexisting health conditions were excluded to reduce confounding by aging- or disease-related physiological changes, such as perimenopause. Articles which studied results in hypoxic conditions and that included women without stratification of results by sex were also omitted. Supplements administered concurrent with additional food or beverage, such as in the context of an energy drink, were not excluded. In total, 34 relevant studies were identified and included in this review. Unless otherwise specified, the results discussed below occurred in studies using only women as participants.

Results

It should be noted that the studies included in this review are limited in sample size and lack procedures to control for the confounding effects of the menstrual cycle, contributing to increased variance in study results. For this reason, a lack of significant difference in the results of presented studies does not necessarily mean that the relevant dietary supplements have no effect, but points to the need for larger sample sizes and better design control (e.g., hormones) in order to make conclusions based on stronger statistical power.

Beta-Alanine

Of the three supplements covered in this review, beta-alanine stands out as the one with the least evidence regarding female athletic performance. Only nine studies report results in women, while over 60 studies have been reported for men. Stout et al. (2007) investigated the effects of chronic beta-alanine supplementation on several measures of aerobic performance and found meaningful improvement in ventilatory threshold (VT) and time-to-exhaustion (TTE), but no improvement in maximal oxygen consumption (VO2max). The 22 women in this study were given 3.2 g of beta-alanine per day for Days 1–7, and 6.4 g/day for Days 8–28, which, relative to body weight, is a high dose (86 mg·kg−1·day−1) compared with those found efficacious in previous studies in men (64 mg·kg−1·day−1) (Stout et al., 2007). Given such a high dose, increases in muscle carnosine were assumed, but were not measured for confirmation. A study of 4.8 g/day chronic beta-alanine supplementation in moderately trained women found nonsignificant increases in VT (p = .11), TTE (p = .83), and VO2max (p = .47), in a treadmill graded exercise test and 40-min run at 70% peak velocity (n= 24) (Smith et al., 2012). Clinical inferences of magnitude suggest that there is likely an ergogenic effect of beta-alanine on TTE, despite the lack of statistical significance. A study by Smith-Ryan et al. (2012), however, found no significant improvements in TTE (p = .48), nor in critical velocity (p = .78) or anaerobic running capacity (p = .78), in a study of the effect of 4.8 g/day beta-alanine on high-velocity intermittent running in recreationally active women or men (n = 50).

Walter et al. (2010) sought to elucidate the combinatory effects of high-intensity interval training with beta-alanine supplementation (6 g/day for Weeks 1–3 and 3 g/day for Weeks 5–7) (n = 44). The results of this study showed that VO2peak (the highest rate of oxygen consumption attained by a subject during an exercise test, though not necessarily the subjects’ physiologically attainable maximum [VO2max]) and power output at VT increased similarly with and without beta-alanine supplementation. This suggests that the physiological adaptations to high-intensity interval training leave no room for additive ergogenic effects of beta-alanine. As such, further study is needed to determine if beta-alanine’s efficacy is perhaps activity-specific. Kresta et al. (2014) investigated the effects of both acute and chronic supplementation of 1 g/kg beta-alanine, alone and in combination with creatine monohydrate, on aerobic and anaerobic performance measures in recreationally active college women (n = 32). The VO2peak, VT, TTE, and total work were measured during a cycle ergometer graded exercise test and no significant differences occurred for any group (p = .20, .44, .24, respectively). The lack of change in muscle carnosine combined with the lack of meaningful increase in performance suggests either that the supplementation protocol may have been insufficient or that some other factor may have affected the metabolism of beta-alanine in these women.

The previous two studies also measured anaerobic performance in the form of peak power at VT (Walter et al., 2010), as well as peak and mean power (Kresta et al., 2014). Neither study found significant effects of beta-alanine on these power outputs (p > .05 for all measures), though the same considerations for dosage and specificity must be taken. Outlaw et al. (2016) investigated the combined effects of beta-alanine and resistance training in a novice population of college-aged females. After 8 weeks of training 4 days/week consuming 3.4 g beta-alanine before training sessions, there were improvements in mean power and muscular endurance. However, such improvements were similar in supplemented and nonsupplemented groups, suggesting that the adaptations came from the resistance training alone with no additive effects of beta-alanine. Ribeiro et al. (2020) studied chronic supplementation with 6.4 g/day of beta-alanine in elite international-level female soccer athletes, which did not lead to any significant improvement in 20-m sprint time (p = .25), repeated sprint ability (p = .67), or recovery (p = .07) (n = 24). In amateur female soccer athletes, a lesser dose of 4.8 g/day beta-alanine did lead to an enhanced improvement in repeated sprint ability, as well as shuttle run and countermovement jump performance, when added to a plyometric training regimen (Rosas et al., 2017). Looking directly at anaerobic performance, Glenn et al. (2015) investigated the acute effects of 1.6 g of beta-alanine ingested 30 min before exercise on mean and peak power, heart rate (HR), blood lactate (BLa), and rate of perceived exertion (RPE) in female cyclists (n = 12). There was no significant change in absolute or relative mean power or peak work, and no effect on HR or BLa by beta-alanine (p > .05 for all measures). The women supplemented with beta-alanine did, however, report significantly lower RPE than the placebo group (p < .001). Smith et al. (2012) also reported significantly lower RPE (p = .03) in women undergoing aerobic testing with beta-alanine, while Kresta et al. (2014) reported improved rate of fatigue in anaerobic testing (p = .04). This suggests that while the overarching outcomes of many female beta-alanine studies are null in terms of direct exercise outputs, beta-alanine may serve as an ergogenic aid by delaying the onset of both anaerobic and aerobic fatigue to allow for longer or more intense training sessions which, in turn, provide greater physiological adaptation for subsequent performance.

Caffeine

As one of the most popular ergogenic aids worldwide, caffeine is the most studied of these three relevant supplements. However, the caffeine studies undertaken in women fail to provide consensus on sex-specific outcomes and recommended intake levels.

Norum et al. (2020) investigated the effects of acute supplementation of 4 mg/kg caffeine on strength and power in resistance-trained women and found significant improvements, while Goldstein, Jacobs et al. (2010) found similar benefits to strength, but no notable improvements in endurance, with 6 mg/kg caffeine supplementation. Using the same 6 mg/kg dosage, Ali et al. (2016) found that evening supplementation with caffeine in female team-sport athletes improved eccentric knee flexor performance both immediately after and the day after consumption. This study did not find a notable difference in strength, nor in peak power or concentric power.

On the other hand, a study in elite female volleyball players showed improvement for volleyball-specific performance measures, such as standing and jumping spike velocity, hand grip, jump height, and agility with 3 mg/kg caffeine ingested 60 min prior to exercise (Pérez-López et al., 2015). Simulated game performance improved and the number of errors per game were reduced in the supplemented group, suggesting that caffeine is meaningfully ergogenic for volleyball and its performance actions. Looking at floorball-specific motor tasks, Krasňanová et al. (2014) found no notable improvement in shooting accuracy, standing long jump, or performance in the 6 × 9 m shuttle run in women supplemented with an average of 4.4 mg/kg caffeine (n = 14). There was a significant improvement in the average completion time of the longer, 6 × 40 m shuttle run (p < .01), which has been shown to reliably reflect power and anaerobic capacity (Baker et al., 1993). There was a large SD in the results, however, which may point to effects of skill or physiological responder status (i.e., level of discernable effect on an individual) on the efficacy of caffeine. Stojanović et al. (2019) investigated the effect of 3 mg/kg caffeine on the anaerobic performance of female professional basketball players (n = 10). Significant increases in 10 m (p = .05) and 20 m (p = .04) sprint times and a moderate decrease in RPE (p = .04) were reported in the supplemented group, along with nonstatistically significant improvements in squat jump (p = .08), agility (p = .12), and suicide run (p = .28) performance. Like Krasňanová et al., the authors found that the response to caffeine was noticably heterogeneous, supporting the possibility of female individuals being “responders” or “non-responders” to caffeine. However, it should be noted that heterogeneity of response does not itself indicate that there are responders and nonresponders for measured outcomes (Atkinson & Batterham, 2015).

Commercial energy drinks are a marketed source of preworkout caffeine, leading researchers to investigate the efficacy of their specific composition and caffeine dosage. Del Coso et al. (2013) supplemented female “rugby sevens” players with 3 mg/kg caffeine in the form of a commercial energy drink. No effect of caffeine was seen on maximal running speed in sprint testing, but total leg muscle power in 15-s maximal jump testing and average running pace during simulated game play improved, showing some ergogenic benefits of the commercial drinks in this population of female rugby athletes. Female soccer players who supplemented with 3 mg/kg caffeine from a commercial energy drink also saw improvements in mean jump height and peak power in a jump test, as well as an increased number and length of sprint bouts per game in simulated game play (Lara et al., 2014). Unlike the female rugby players, these supplemented female soccer players had increased average peak speed and maximal speed in a sprint test, though this did not translate to any significant difference in maximal speed (p = .25) during game play (n = 18). Astorino et al. (2012) supplemented female soccer payers with the popular energy drink Red Bull®, which contains only 1.3 mg/kg caffeine, before testing repeated sprint performance. There were no beneficial effects in the supplemented group on sprint time, RPE, or HR, which, in the absence of other clear confounding variables and compared with the positive results of higher dose studies, suggests that the 1.3 mg/kg caffeine provided in one serving of Red Bull® is an insufficient dose to be effective in female athletes. Given the popularity of energy drinks, it is reasonable to assume that nonathletes also consume them with the intention of reaping their advertised benefit. In a study of untrained females (n = 32) given commercial energy drinks at a dose of 4 ml/kg, no significant difference was seen between caffeine and placebo groups for TTE (p < .16), HR (p = .72), blood lactate (p = .85), or VO2max (p < .15) (Al-Fares et al., 2015). Importantly, the dosage of energy drink was measured in milliliters of drink per kilogram body weight. The authors do not clearly specify the dosage of caffeine that one would consume with this volume, making it difficult to objectively compare this study with others mentioned.

Also investigating commercial products, Tinsley et al. (2017) compared the effects of both caffeinated and noncaffeinated commercial energy drinks on muscular force production in women and men (n = 21). The caffeinated energy drink contained 3.6 mg/kg caffeine, and the rest of the ingredients varied between the two drinks, save for a consistent 6 g citrulline malate in both beverages. The increases in strength seen in the results of a three rep max force production test in both men and women were statistically nonsignificant (p = .57). However, the differences between men and women in the improvements that did occur were much more pronounced in the noncaffeinated group (0%–1.5% increase for women, 9% for men) compared with the caffeinated group (5%–11% increase for women and men). Skinner et al. (2019) examined the sex-based differences in 3 mg/kg caffeine supplementation on endurance cycling performance and found a significant increase in cycling time-trial (TT) performance with caffeine (p < .001), but no significant difference between sexes (p = .98) (n = 27). Notably, postexercise plasma concentrations of caffeine were significantly higher in women (p < .001) than in men, with only women experiencing significant pre- to postexercise increases in plasma caffeine concentration (p < .01). While both studies appear to demonstrate no difference in caffeine efficacy between sexes, the more pronounced increase in plasma caffeine concentration and greater benefit to strength performance compared with noncaffeinated energy drinks may support the idea of different metabolic responses to caffeine in women compared with men. This possibility is exemplified by Potgieter et al. (2018), who found a greater magnitude of response to 6 mg/kg caffeine in triathlon performance in men compared with women. Importantly, for the aforementioned studies of commercial energy drinks, any observed effects may be attributed to other ingredients in the beverage mixtures and not to caffeine alone.

Further evidence of sex-based responses to caffeine is illustrated by Chen et al. (2019) in their study on caffeine’s impact on delayed-onset muscle soreness and exercise-induced muscle damage in both male and female college athletes (n = 20). One hour after ingestion of 6 mg/kg caffeine, men saw a significantly greater decrease in reported delayed-onset muscle soreness compared with women (p < .01). This study also showed a reverse correlation between the reduction in delayed-onset muscle soreness and the restoration of power output after exercise-induced muscle damage in men, but no such correlation was found in women. However, the small sample size (n = 10 F, 10 M) and variance related to interindividual differences in hormone levels may have contributed to the lack of detectable correlation in women. Motl et al. (2006) found a significant reduction in midworkout pain perception (p = .001) in moderately trained college women given either 5 or 10 mg/kg caffeine before exercise (n = 11). Decreased perception of pain midworkout could translate to longer or harder training sessions in women, leading to greater physiological adaptation to training. The similar efficacy of the 5 and 10 mg/kg doses suggests that there may be a threshold after which the dose–response effect of caffeine plateaus.

Nitrate

The review by Wickham and Spriet (2019) makes clear the sex-based disparity in research on dietary nitrate supplementation. Only 10 studies investigating the effects of dietary nitrate in women were found for use in this review. Among these 10 studies, conclusions are mixed.

Investigating aerobic effects of nitrate on an exercise TT performance, Peeling et al. (2015) saw a significant 1.7% increase in 50-m TT performance (p = .01) in elite female kayak athletes after an acute dose of ∼9.6 mmol of dietary nitrate in the form of beetroot juice 2 hr prior to exercise (n = 5). This study, however, did not measure plasma nitrate or NO2 to verify that positive changes in performance could be due to nitrate pathways. Pospieszna et al. (2016) found significant improvement in swimming TTs (p < .01) in female university-level swimmers after chronic 8-day beetroot juice supplementation with only 5.1 mmol of dietary nitrate per day (n = 11).

Three additional TT studies saw no marked effect of dietary nitrate on performance. In recreationally active females supplemented both acutely and chronically with ∼26 mmol nitrate per day, there was no difference in cycling TT performance or mean submaximal VO2, despite an increase in plasma nitrate and NO2 (Wickham et al., 2019). In well-trained competitive female cyclists singularly or concurrently supplemented with ∼7.3 mmol nitrate 2.5 hr before exercise and 5 mg/kg caffeine 1 hour before exercise, TT performance was not altered by NO3, regardless of caffeine status (Glaister et al., 2015). A similar study in elite Olympic-level cyclists showed no effect on TT performance with two 8.4 mmol doses of nitrate both 10–12 hr and ∼2 hr prior to exercise. In those athletes also given 3 mg/kg caffeine, there were no additive benefits of nitrate compared with caffeine supplementation alone (Lane et al., 2014). The three studies which found no effect of dietary nitrate supplementation via beetroot juice all investigated cycling performance outcomes, while the two studies which found positive results investigated swimming and kayaking, both upper body heavy exercises. This may suggest that dietary nitrate is more efficacious in upper body compared with lower body endurance sports.

It has been suggested that dietary nitrate supplementation has greater efficacy in untrained and moderately trained individuals compared with highly trained athletes (Hoon et al., 2013; Senefeld et al., 2020). While reviews which make this suggestion do so using primarily male data, the only mentionable study in untrained women, Fernandes de Castro et al. (2018), found no significant improvement in 3-km race performance (p = .77) with acute prerace supplementation with 8.4 mmol NO3(n = 8). Despite the contradiction presented by this one study in women, replication of results is needed before making conclusions about training status and efficacy in women specifically.

One unique study investigated the effects of acute nitrate supplementation on self-regulated exercise performance outcomes using an “RPE-clamp” design at a set RPE of 13, equating to the perception of “somewhat hard” work (Rienks et al., 2015). In this study, 10 recreationally active women were supplemented with 12.9 mmol nitrate, 150 min prior to 30 min of exercise on a cycle ergometer, the middle 20 min occurring at an RPE of 13. Dietary nitrate did not have an effect on total VO2 or rate of mechanical work compared with placebo. The self-regulated nature of this protocol intended to mimic a typical independently paced aerobic training session for a recreational or moderately trained athlete. The lack of distinctions among the performance outcomes of this study suggests that dietary nitrate may not have notable benefits to the quality of daily training in female athletes, especially when sessions are self-regulated and not dictated by coaches or trainers.

When examining anaerobic performance, Jonvik et al. (2018) found no significant increase in swimming sprint performance (p = .18), despite an increase in plasma NO3, in elite female water polo players after 6 days of chronic beetroot juice supplementation totaling ~12 mmol nitrate per day (n = 14). Similarly, 6 mmol nitrate supplemented 3 hr before exercise had no effect on repeated sprint ability in amateur female team-sport athletes despite increases in plasma nitrate concentration (Buck et al., 2015). In the previously discussed study of elite Olympic level cyclists, Lane et al. (2014) saw no effect of beetroot juice on power output, either alone or in combination with caffeine. Contrarily, the study by Pospieszna et al. (2016) found more pronounced changes in anaerobic sprint swimming performance compared with aerobic TTs results. While this outcome could suggest that dietary nitrate provides stronger ergogenic benefits in anaerobic activity versus aerobic activity, the small sample size (n = 11) and lack of similar outcomes in other female studies necessitates further research on both aerobic and anaerobic performance.

Casado et al. (2021) investigated the effect of nitrate supplementation on the performance of amateur runners and the extent to which sex may play a role in such effects. 10 women and 14 men were given ~12.8 mmol nitrate 2.5 hr before a 2-km TT. There was a significant improvement in TT performance (p = .002) and a reduction in RPE (p = .01); however, the magnitude of the response was similar in men and women. This study suggests that there may not be sex-related divergences in response to dietary nitrate, and points to the need for future sex-stratified research into the ergogenic benefits of nitrate supplementation to create direct comparison between men and women.

Discussion

Despite their frequent use by recreational, amateur competitive, and professional athletes, beta-alanine, caffeine, and nitrate are ergogenic dietary supplements which lack strong evidence for efficacy in women, specifically. A summary of the findings from the 34 studies in this review is found in Table 1. Collectively, the studies of beta-alanine in women suggest that while the supplement may not have any direct additive effects on the physiological adaptations to exercise training, its buffering capacity may serve to lower RPE, thus allowing longer exercise sessions and enhanced training adaptations. The results of caffeine supplementation in women are mixed, with the possibility of pain reduction, improved anaerobic performance, and aerobic performance all evidenced in individual studies. There may be a difference in caffeine’s efficacy based on physiological responder status, with some individuals reacting more strongly than others, as well as a dose–response relationship in which only a certain range of caffeine dosed in relation to body weight may prove effective. Finally, the limited female-based studies of nitrate provide little consensus on efficacy, though the possibilities of distinct effects on upper and lower body-specific exercises are presented. In sum, the research in women thus far has not led to convincing conclusions regarding sex-based differences in supplement efficacy, and the existing studies have limitations which impact their applicability.

Table 1

Effects of Dietary Beta-Alanine, Caffeine, and Nitrate Supplementation on Exercise Performance in Studies Involving Women

ReferencesPopulationNumber, age, and sex of participantsSupplement dosageExercise protocolMain outcomesPower analysis
Beta-alanine
 Glenn et al. (2015)Trained, competitively active cyclists (average = 3.9 y training)

All participants in luteal phase of menstrual cycle (48 hr postcessation of menses)
n = 12 F

Age = 26.6 ± 0.3
1.6 g3 × Wingate trials with 2-min active rest between↑ RPE

↔ Mean power, peak power, HR, BLa
 Kresta et al. (2014)Moderately active (≥30 min 3×/week) adultsn = 32 F

Age = 21.5 ± 2.8
0.1 g/kg days 1–28GXT (cycle ergometer) and 2 × 30-s Wingate tests at work rate of 7.5 J·kg−1·rev−1 with 3-min rest between↔ Mean power, peak power, total work

↑ Rate of fatigue
 Outlaw et al. (2016)Untrained adultsn = 16 F

Age = 21 ± 2.2
3.4 g before 4× weekly trainingBruce protocol (treadmill), Wingate test, 1RM bench press, 65% 1RM leg press to failure, vertical jump, and standing broad jump tests↔ Peak power, max strength, bench press reps to failure, VO2, TTE

↑ Vertical jump, standing broad jump, leg press RTF
 Ribeiro et al. (2020)International level elite soccer athletesn = 24 F

Age = 18 ± 1
1.6 g 4×/day (6.4 g total)Yo-Yo IR1, RAST, and 20-m maximal sprint test↔ Repeated-sprint ability, 20-m sprint time, Yo-Yo IR1
 Rosas et al. (2017)Amateur soccer athletesn = 25 F

Age = 23.7 ± 2.4
0.8 g 6×/day (4.8 g total)Squat jump, countermovement jump, 20-m sprint test, RAST, 40-cm drop jump reactive strength index, peak jump power, change-of-direction speed (Illinois test), 20-m multistage shuttle run, and 60-s countermovement jump↑ 60-s countermovement jump power, repeated sprint ability, shuttle run

↔ Change of direction speed, squat jump, peak jump power, reactive strength
95% power, alpha = .01
 Smith et al. (2012)Moderately trained (3–7×/week) adultsn = 24 F

Age = 21.7 ± 2.1
1.6 g 3×/day Days 1–28 (4.8 g total)GXT (treadmill) and 40-min treadmill run at 70% peak velocity↔ VO2max, VT, TTE, HR

↑ RPE
 Smith-Ryan et al. (2012)Recreationally active (1–5 hr/week) adultsn = 24 F, 26 M

Age = 21.7 ± 2.1 (F), 22.0 ± 3.3 (M)
1.6 g 3×/day (4.8 g total)3 × run to exhaustion at 100% PV, 90% PV, and 110% PV with 15 min of rest between each bout↔ TTE, critical velocity, anaerobic running capacity
 Stout et al. (2007)Not explicitly mentioned—assumption is either untrained or recreationally active adultsn = 22 F

Age = 27.4 ± 6.1
3.2 g/day days 1–7

6.4 g/day days 8–28
GXT (cycle ergometer)↑ VT, working capacity, TTE

↔ VO2max
 Walter et al. (2010)Recreationally active (1–5 hr/week) adultsn = 44 F

Age = 21.8 ± 3.7
1.5 g BA 4×/day weeks 1–4 (6 g total)

1.5 g BA 2×/day weeks 5–7 (3 g total)
GXT (cycle ergometer) Weeks 0, 4, and 8.

30 min of HIIT on the bike 3× per week, Weeks 1–3 and 5–7
↔ VO2peak, power at VT
Caffeine
 Al-Fares et al. (2015)Untrained (not regularly exercising) adultsn = 32 F

Age = 19.9 ± 0.8
4 ml/kg BWBruce protocol (treadmill)↔ TTE, VO2max, HR, BLa

↑ BP
90% power, alpha = .05
 Ali et al. (2016)Varied: recreationally active adults— > internationally competitive athletes

All on oral contraceptives, with exercise testing occurring on same days of cycle
n = 10 F

Age = 24 ± 4
6 mg/kg BW2 × 15 min blocks of intermittent treadmill running protocol

Eccentric, concentric, and isometric contractions of knee flexors and extensors (isokinetic dynamometer)
↑ Knee flexor eccentric toque

↔ Knee flexor concentric torque, knee extensor eccentric + concentric torque, isometric strength, jump height, power, VO2, HR
 Astorino et al. (2012)College soccer athletes (~12 hr training/week)n = 15 F

Age = 19.5 ± 1.1
1.3 mg/kg BW3 × 8 “all-out” t-test sprints w/5-min rest between sets↔ Sprint performance, RPE, HR
 Chen et al. (2019)Division 1 college athletes

No oral contraceptive use, all exercise testing in early follicular phase of menstrual cycle
n = 10 F, 10 M

Age = 20.4 ± 1.2 (F), 21.1 ± 2.1 (M)
6 mg/kg BWDownhill running protocol to induced DOMS/EIMD

24 and 48 hr after EIMD, muscle performance measured using knee flexor/extensor peak power on a dynamometer
↑ Muscle performance, M and F

↑ DOMS, M > F

↑ EIMD, M only
 Del coso et al. (2013)National-level rugby “sevens” athletesn = 16 F

Age = 23 ± 2.0
3 mg/kg BW6 × 30 m sprint test, three rugby competition games with 15-min rest between games, 15-s jump test↔ Max running speed, HRmax

↑ Leg muscle power, mean running pace
 Goldstein, Jacobs, et al., (2010)Resistance trained (3–5 days/week)n = 15 F

Age = 24.6 ± 6.9
6 mg/kg BW1RM barbell bench press test and RTF at 60% of 1RM↑ 1RM

↔ RTF, HR, BP
 Krasňanová et al. (2014)Competitive floorball team athletesn = 14 F

Age = 23.3 ± 5.3
4.4 mg/kg BWStanding long jump, 6 × 9 m shuttle run, reaction speed test, shooting accuracy test, and 6 × 40 m shuttle run↑ 6 × 40m shuttle run time

↔ 9 × 40 m shuttle run time, leg muscle power, shooting accuracy, and reaction speed
 Lara et al. (2014)Competitive soccer athletes (6–8 hr training/week)n = 18 F

Age = 21 ± 2.0
3 mg/kg BWJump test, 7 × 30 m max running speed test, and a simulated soccer game (2 × 40 min halves)↔ Max sprint speed

↑ Jump height, peak power, peak sprint speed, number, and length of sprints
 Motl et al. (2006)Adults of average fitness (not sedentary but not trained)

No oral contraceptive use, all exercise testing occurred in self-reported follicular phase of menstrual cycle
n = 11 F

Age = 19.5 ± 1.1
5 mg/kg BW

10 mg/kg BW
30-min cycling at 60% VO2peak↔ Power, HR, SBP, VO2

↑ Pain intensity ratings
80% power, alpha = .05
 Norum et al. (2020)Resistance-trained (2–3 days/week) adults, all subjects White

All exercise testing occurred in early follicular phase of menstrual cycle, some participants using oral contraceptives and some not
n = 15 F

Age = 29.8 ± 5.5
4 mg/kg BW1RM, RTF at 60% of 1RM, isometric knee extensions, countermovement jump↑ 1RM, RTF, MVC torque, countermovement jump, power

↔ Force, muscle activation
80% power, alpha = .05
 Pérez-López et al. (2015)National-level elite volleyball athletes

Exercise testing occurred in follicular phase (n = 4) and luteal phase (n = 9)
n = 13 F

Age = 25.2 ± 4.8
3 mg/kg BWStanding spike and jump spike, maximal spike jump, squat/countermovement/block jumps, max manual dynamometry (hand grip), and agility t test (all ×2 with 1 min between reps and 3 min between tests)↑ Handgrip, max ball spike velocity, jump height, agility t test, HRmean, HRmax
 Potgieter et al. (2018)Competitive triathletes, all subjects Whiten = 12 F, 14 M

Age = 37.2 ± 11.6 (F), 38.4 ± 10.0 (M)
6 mg/kg BWTriathlon↑ Overall time M only, swim time

↔ RPE
 Skinner et al. (2019)Competitive cyclists/triathletes

All participants using oral contraceptives, all exercise testing occurred in luteal phase of menstrual cycle
n = 11 F, 16 M

Age = 29.7 ± 5.3 (F), 32.6 ± 8.3 (M)
3 mg/kg BW2 × cycling TT↔ RPE

↑ Cycling performance, HR
80% power, alpha = .05
 Stojanović et al. (2019)Professional basketball athletes

All exercise testing occurred in luteal phase of menstrual cycle
n = 10 F

Age = 20.2 ± 3.9
3 mg/kg BWJump test with and without arm swing, squat jump, lane agility drill, 20-m sprints with and without dribbling, and 140-m suicide run↑ 10 and 20 m sprint times

↔ Jump height, repeated sprint performance, lane agility drill
80% power, alpha = .05
 Tinsley et al. (2017)Resistance-trained (7.5 ± 3.9 hr/week) adults

Subjects included 11 White, six Hispanic, three Asian, and one African American
n = 12 F, 9 M

Age = 21.5 ± 2.0 (F), 20.7 ± 2.8 (M)
3.6 mg/kg BWThree rep max force production test and 5 × 6 isokinetic squats at perceived maximal effort↔ Concentric force, eccentric force, RPE, fatigue80% power, alpha = .05
Nitrate
 Buck et al. (2015)Amateur team-sport athletes (195 ± 42 min play/week) n = 9 participants using oral contraceptives, n = 4 participants not using oral contraceptivesn = 13 F

Age = 25.5 ± 1.9
6 mmolSTGC consisting of four 15-min quarters, 3 × RSA↔ Sprint times, RPE, HR
 Casado et al. (2021)Amateur runnersn = 10 F, 14 M

Age = 36.6 ± 8.2 (F), 38.7 ± 9.2 (M)
12.8 mmol2-km running TT↑ RPE M and F

↑ TT M and F

↔ BLa
 Fernandes de Castro et al. (2018)Untrained adultsn = 8 F

Age = 30 ± 5.7
8.4 mmol2 × 3-km running TT↔ TT performance, HRmax, BLa

↑ RPE
 Glaister et al. (2015)Competitive cyclists/triathletes (10.7 ± 2.2 hr training/week)n = 14 F

Age = 31 ± 7
∼7.3 mmol4 × 20-km cycling TT↔ VO2, RPE, HR, BLa
 Jonvik et al. (2018)National-level water polo athletesn = 14 F

Age = 22 ± 4
∼12 mmolIntermittent swim sprint test 4 × 4 15-m sprints w/30-s active rest between sets↔ Swim sprint performance, dynamic apnea distance
 Lane et al. (2014)International-level competitive cyclists/triathletesn = 12 F, 12 M

Age = 28 ± 6 (F),

31 ± 7 (M)
8.4 mmol × 2 (16.8 mmol total)Cycling TT (F: 29.35 km, M: 43.83 km)↔ TT performance, mean power
 Peeling et al. (2015)International-(Olympic-) level kayak athletesn = 5 F

Age = 25 ± 2.8
∼9.6 mmol50-m Kayak Paddle TT↑ TT performance
 Pospieszna et al. (2016)University league swimmers (three practices per week)n = 11 F

Age = 20.9 ± 1.3
5.1 mmol/day × 8 days6 × 50-m maximal swim sprint, 800-m continuous swim↑ Swim sprint performance, endurance TT

↔ HR, BP
 Rienks et al. (2015)Recreationally active (VO2peak ~35 ml·kg·−1·min−1) adultsn = 10 F

Age = 25 ± 3
12.9 mmol“RPE Clamp” Protocol at RPE of 13↑ VO2 at fixed work rate

↔ Total VO2, total work
 Wickham et al. (2019)Recreationally active (VO2peak 35–35 ml·kg·−1min−1, 4.5 ± 1.5 hr exercise/week) adults

All participants using hormonal contraceptives
Study 1: O2 and performance

n = 12 F

Age = 23 ± 1

Study 2: Contractile properties

n = 12 F (n = 7 from Study 1) Age = 22 ± 2
∼26 mmol nitrate × 1 day and × 8 daysStudy 1: Submaximal cycling protocol, 4 kJ/kg BW cycling TT

Study 2: 3 × MVC of plantar flexors with 3-min rest between tests, electric current stimulation to achieve 30% MVC at 100 Hz
↔ TT performance, VO2

↑ Plantar flexor torque

Note. The “↑” indicates improvement and “↔” indicates no change. 1RM = one-repetition maximum; BA = beta-alanine; BLa = blood lactate; BP = blood pressure; BW = body weight; DOMS = delayed onset muscle soreness; EIMD = exercise-induced muscle damage; GXT = graded exercise test; HIIT = high-intensity interval training; HR = heart rate; MVC = maximum voluntary contraction; PV = peak velocity; RAST = running anaerobic sprint test; RPE = rate of perceive exertion; RSA = repeated sprint ability; RTF = repetitions to failure; SBP = systolic BP; STGC = simulated team game circuit; TT = time trial; TTE = time-to-exhaustion; VO2 = oxygen uptake; VO2max = maximal VO2; VO2peak = peak VO2; VT = ventilatory threshold; Yo-Yo IR1 = Yo-Yo Intermittent Recovery Test Level 1.

Limitations of Current Literature

Hormonal Status

Of the 34 studies addressed in this review, all of which included female participants, only 10 (one on beta-alanine, seven on caffeine, and two on nitrate) controlled for menstrual cycle and hormonal status in some way. Menstrual phase in women and hormonal status have been shown to impact exercise metabolism (Devries et al., 2006; Tarnopolsky, 2008), beta-alanine metabolism (Draper et al., 2018), caffeine clearance rates (Lane et al., 1992), and plasma NO3: NO2 ratios (Chatterjee & Mukhopadhyay, 2015). Of the 10 studies which took into account the cyclic fluctuations of sex hormones, there was no uniformity in which phase of menstruation was controlled. Studies occurred with and without hormonal contraceptives, in luteal and follicular phases, and in some cases included multiple phases and contraceptive statuses in one testing group without stratification of results based on these differences. Conducting studies on women is the first step to mitigating the gap in research between men and women regarding exercise physiology and nutritional supplementation (Elliott-Sale et al., 2021). However, by ignoring a major physiological reason why “women are not small men,” this research limits itself in generalizability to the female population (Dr. S. Sims, quote—TED talk, 2019).

In additional to the intraindividual variation in hormonal status and metabolism throughout the menstrual cycle, there is also potential for interindividual differences in physiological response to supplementation and exercise protocols that can influence the generalizability of results. Factors influencing interindividual variation include genetics, sleep, stress, and training status and modality (Mann et al., 2014). These factors affect outcome variation in both men and women and must be noted as a limitation to all nutrition and exercise metabolism studies, including those in this review.

Additional Gaps and Future Directions

While lack of clear consideration for hormonal status is the primary limitation in many of these studies, several other notable gaps exist. The studies conducted in women are often statistically underpowered; the average N value presented in Table 1 is 16. In addition to small sample size, which can undermine the validity and generalizability of results, the inclusion of studies on mixed-substance supplements increases the risk of confounding by other active ingredients rather than the specific supplement in question. This is particularly true of caffeinated energy drinks, whose common ingredients, such as citrulline malate and taurine are suggested, though not widely proven, to enhance performance by their own mechanisms (Gough et al., 2021; Kurtz et al., 2021).

In seeking to understand the differences in performance and dietary interactions between men and women, it is important to inform such variations using as many characteristics as possible. This includes not only physiological differences, such as hormonal status, but also lifestyle and demographic characteristics that may play a role in these variances. While any data in women are a step in the right direction, the participants in these studies are largely homogenous in terms of race and fall within a narrow mean age group. Racial differences have been identified in the metabolism of micronutrients and their relationships to other metabolic biomarkers, including endogenous hormones, such as Vitamin D (Gutiérrez et al., 2011; Suarez & Schramm-Sapyta, 2014). In relation to age, declines in the major sex hormones estrogen and progesterone begins as early as the 30s for women (Hale & Burger, 2009). Though literature reported with participants under 40 years of age were included in this review, most of these studies (30/34) were conducted in women of mean age below 30 years. By not commonly including women past the age of 30 years, researchers could be missing other important relationships between women and performance-enhancing supplements that exist through hormonal interactions or lack thereof.

Unlike race and age, the training status of participating women in these studies was heterogeneous, varying from untrained to internationally competitive athletes. With only a few studies available, it is difficult to ascertain any replicable relationship between training status and degree of efficacy in women for the three supplements herein. However, studies in men have suggested relationships between training status and response to ergogenic aids, specifically nitrate (Hoon et al., 2013; Senefeld et al., 2020).

Though literature on women in relation to nutrition for exercise and sport is limited, there is an emerging focus on expanding the bodies of research to minimize the sex-based gaps in this field. A recent publication by Elliott-Sale et al. (2021) presents considerations for exercise-based studies in women, including specifications for participant selection, nomenclature, and experimental design. In the call for the increased inclusion of women in ergogenic dietary supplement study protocols, these methodological recommendations apply. At this moment, there is not enough literature available to clearly elucidate patterns of effect magnitude and age, race, or training status. While the priority of continued research focuses on understanding and accurately controlling for major hormonal differences in women, it is prudent for researchers to begin to diversify their study populations to include women of greater age ranges, various races, and specific training statuses as the field continues to expand to better understand all potential variables contributing to the female response to supplementation.

Conclusion

Despite growing awareness of the need for female-specific exercise and nutrition research, the body of literature on the common ergogenic aids beta-alanine, caffeine, and nitrate remains limited. Practical application of the studies discussed herein remains difficult as potential differences in hormonal status both between men and women, and within women across the menstrual cycle, are not yet supported by high-quality evidence in relation to exercise performance and thus continue to limit application of outcomes to generalized guidelines for women in sport (McNulty et al., 2020). While past reviews have highlighted the efficacy of beta-alanine, caffeine, and nitrates individually, this review is the first to consolidate the relevant studies of these three common ergogenic aids and discuss outcomes and implications specifically for women (see Figure 1). Further research in women is necessary to continue to illuminate potential sex-specific dosage, timing, and general efficacy guidelines for beta-alanine, caffeine, and nitrate. As this research continues, the methodological considerations for including women in exercise protocols outlined by Elliott-Sale et al. (2021) should be applied as the standard of practice. These three supplements continue to be used by athletes and are recognized by professional organizations as effective ergogenic aids which enhances the likelihood of their continued popularity.

Figure 1
Figure 1

—Summary of mechanisms and effects for beta-alanine, caffeine, and nitrate in women. RPE = rate of perceive exertion.

Citation: International Journal of Sport Nutrition and Exercise Metabolism 32, 4; 10.1123/ijsnem.2021-0176

Acknowledgments

All authors conceptualized the study. M.J. Murphy assisted in investigating and the writing the original draft. The study was reviewed and edited by B.R. Rushing, S.J. Summer, and A.C. Hackney. A.C. Hackney also helped in the visualization of the study.

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Hackney (ach@email.unc.edu) is corresponding author.

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  • Figure 1

    —Summary of mechanisms and effects for beta-alanine, caffeine, and nitrate in women. RPE = rate of perceive exertion.

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