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

Molly J. Murphy

The University of North Carolina at Chapel Hill

Gillings School of Global Public Health

A paper submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of
the requirements for the degree of Master of Public Health in the Department of Nutrition

May 4, 2021

Approved by:
Anthony C. Hackney
(Paper Advisor)

Date: May 3, 2021

1
Dietary Supplements for Athletic Performance in Females

ABSTRACT

Beta-alanine, caffeine, and nitrate are dietary supplements generally recognized by the sport

and exercise science community as being evidence-based ergogenic aids to performance. The evidence

supporting the efficacy of these supplements, however, is greatly skewed due to research being

conducted primarily in males. The physiological difference between men and women, most notably the

differences in sex hormones and hormonal changes during the menstrual cycle, makes generalizing male

data to the female athlete inappropriate, and potentially harmful to females. This narrative review

outlines the studies conducted in women to date regarding the efficacy of beta-alanine, caffeine, and

nitrate supplementation for performance enhancement. Only 9 studies on beta-alanine, 15 studies on

caffeine, and 10 studies on nitrate in healthy women under the age of 40 conducted in normoxia

conditions were identified as relevant to this research question. Evidence suggests that beta-alanine

may hold the potential to 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 play a role in the benefits of caffeine, 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, considerations for hormonal status or racial/ethnic differences among

women is currently lacking in the literature. This gap in sex-based knowledge necessitates further

research on these ergogenic supplements in women.

KEY WORDS Sport, Female, Physiology, Training, Competition, Adaptation

Dietary Supplements for Athletic Performance in Females

INTRODUCTION

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 or endurance, though the range of supplements on the

market far exceeds the number whose efficacy have been adequately studied and reported (Molinero

and Márquez 2009; Maughan et al. 2018). It is estimated that the rate of supplement consumption in

athletes is higher than that of the general US population, with the highest use in elite athletes compared

to non-elite athletes (Knapik et al. 2016). Studies of college athletes suggest that between 64-89%

regularly use at least one dietary supplement (Osterman et al. 2020; Froiland et al. 2004; Knapik et al.

2016).

The prevalence of 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 comes from studies that have primarily investigated the effect of

these ergogenic aids on the performance outcomes of 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-gender studies in the 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.

Three ergogenic supplements in particular; beta-alanine, caffeine, and nitrate; are identified by the

American College of Sports Medicine as having evidence-based uses in sport nutrition, yet present a

strong disparity in sex-specific research (Thomas et al. 2016a). Physiologically these ergogenic aids work

on different bodily systems and involve varying mechanisms for effect (as discussed in the following

section), and as such are utilized in the training-competitions of a multitude of sporting activities.
Dietary Supplements for Athletic Performance in Females

A study on substance use in NCAA athletes reported that 12.1% of collegiate athletes use

supplemental amino acids for ergogenic purposes (Anon n.d.). Beta-alanine is one such amino acid that

has been studied for its performance enhancing properties. Beta-alanine is a non-essential, non-

proteinogenic amino acid. 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 potentially 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, both

support the amino acid’s role in enhancing exercise performance (Trexler et al. 2015; Thomas et al.

2016b).

Caffeine is one of the most commonly used ergogenic aids in sport, with 28.6% of NCAA athletes

reporting use of caffeine-containing energy drinks (Anon 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 (E. R. Goldstein et al. 2010). Caffeine functions as an

adenosine receptor antagonist which allows for increased release of stimulating neurotransmitters such

as acetylcholine and norepinephrine, leading to enhanced arousal and reduced fatigue. Caffeine is also

proposed to function as an ergogenic aid by promoting fatty acid mobilization and shifting substrate
Dietary Supplements for Athletic Performance in Females

utilization away from glycogen (E. R. Goldstein et al. 2010; Ganio et al. 2009). 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 (Guest et al. 2021; E. R. Goldstein et al. 2010; 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 forms in which inorganic

nitrate (NO3-) is consumed. 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 ATP synthesis to improve mitochondrial efficiency (Lidder and

Webb 2013; Larsen et al. 2011). Nitrate has been shown to be efficacious in enhancing both sustained

sub-maximal aerobic exercise, as well as short-duration high-intensity exercise, though more data is

needed to clarify optimal dosage and timing of supplementation (Maughan et al. 2018; Hoon et al.

2013).

Despite being included positively in the position statements of various sport and nutrition

professional organizations, there is a lack of scientific evidence regarding the efficacy of beta-alanine,

caffeine, and nitrate for improving the exercise performance of women. These three supplements are

prime examples of highly prevalent ergogenic aids that are advertised and used without strong evidence

of efficacy in women. Guidelines for supplementation, though sometimes based on bodyweight, do not

differentiate between men and women, implying that no sex-based differences exist, when in reality this
Dietary Supplements for Athletic Performance in Females

has not been proven (Maughan et al. 2018). In light of these position statements and guidelines, 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. This is

warranted as there are several physiological differences between males and females that may alter the

effect of dietary supplements in each sex. By summarizing the current literature, this review aims to

highlight the disparity in ergogenic research in females and the need 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 databases of PubMed,

SPORTDiscus, Scopus, and Google Scholar. 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 – March 2021. In keeping with the focus of this review, articles that included

participants with a mean age >40 years or those with preexisting health conditions, that studied results

in hypoxic conditions, and that included women without stratification of results by sex were 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

Beta-Alanine

Of the three supplements covered in this review, beta-alanine stands out as the one with the

least evidence in regard to female athletic performance. Only 9 studies report results in women, while
Dietary Supplements for Athletic Performance in Females

over 60 studies have been reported for men. Stout et al. (2007) investigated the effects of chronic (28

day) 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 3.2g of beta-alanine per

day for days 1-7 of the protocol, and 6.4g per day for days 8-28, which, relative to body weight, is a high

dose (86mg/kg/day) compared to those found efficacious in previous studies in men (64 mg/kg/day)

(Stout et al. 2007). Given such a high dose, increases in muscle carnosine were assumed, but were not

measured for confirmation. A study of 4.8g per day chronic (28 day) beta-alanine supplementation in

moderately-trained women found nonsignificant increases in VT and TTE, as well as VO2max, in a

treadmill graded exercise test (GXT) and 40-minute run at 70% peak velocity (Smith et al. 2012). Clinical

inferences of magnitude suggest that there is a very likely ergogenic effect of beta-alanine on TTE,

despite the lack of statistical significance. A study by Smith-Ryan et al. (2012), however, also found no

significant improvements in TTE, nor in critical velocity or anaerobic running capacity, in a study of the

effect of 4.8g/day beta-alanine on high-velocity intermittent running in recreationally active women or

men.

Walter et al. (2010) sought to elucidate the combinatory effects of High Intensity Interval

Training (HIIT) with beta-alanine supplementation (6g/day for weeks 1-3 and 3g/day for weeks 5-7 with

testing on weeks 0, 4, and 8). 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 in a similar manner

with and without beta-alanine supplementation. This suggests that the physiological adaptations to HIIT

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 (28 day) supplementation of 1g/kg beta-alanine, alone and in
Dietary Supplements for Athletic Performance in Females

combination with creatine monohydrate, on aerobic and anaerobic performance measures in

recreationally active college women. In this study muscle carnosine was measured, but no significant

differences were observed across groups. The VO2peak, VT, TTE, and total work were measured during a

cycle ergometer GXT and no significant differences were found for any group. The lack of change in

muscle carnosine combined with the lack of meaningful increase in performance suggests either that

the supplementation protocol for this study 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, though the same considerations for dosage and

specificity must be taken in regards to these outcomes. Outlaw et al. (2016) investigated the combined

effects of beta-alanine supplementation and resistance training in a novice population of college-aged

females. After 8 weeks of training 4 days/week with supplementation of 3.4g beta-alanine immediately

before each training session, there were improvements in mean power and muscular endurance.

However, such improvements were similar in supplemented and non-supplemented groups, suggesting

that these adaptations came from the resistance training alone with no additive effects of beta-alanine.

Ribeiro et al. (2020) studied chronic 21-day supplementation with 6.4g/day of beta-alanine in elite

international-level female soccer athletes, which did not lead to any significant improvement in 20m

sprint time, repeated sprint ability, or recovery. In amateur female soccer athletes, a lesser dose of

4.8g/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

1.6g of beta-alanine ingested 30 minutes prior to exercise on mean and peak power, heart rate (HR),

blood lactate, and rate of perceived exertion (RPE) in female cyclists. There was no significant change in
Dietary Supplements for Athletic Performance in Females

absolute or relative mean power or peak work, and no effect on HR or blood lactate by beta-alanine.

The women supplemented with beta-alanine did, however, report significantly lower RPE than the

placebo group. Smith et al. (2012) also reported significantly lower RPE in the women undergoing

aerobic testing with beta-alanine, while Kresta et al. (2014) reported improved rate of fatigue in

anaerobic testing. 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 world-wide, caffeine is the most commonly studied of

these three relevant supplements. However, the caffeine studies undertaken in women still fail to

provide consensus on sex-specific outcomes and recommended intake levels.

Norum et al. (2020) investigated the effects of acute supplementation of 4mg/kg caffeine on

strength and power in resistance-trained women and found significant improvements, while Goldstein

et al. (2010) found similar benefits to strength, but no notable improvements in endurance, with 6mg/kg

caffeine supplementation. Using the same 6mg/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 were there improvements in peak power or concentric power.

On the other hand, a study in elite female volleyball players showed significant improvement for

volleyball-specific performance measures such as standing and jumping spike velocity, hand grip, jump

height, and agility with 3mg/kg caffeine ingested 60 minutes 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 in regard to volleyball and its
Dietary Supplements for Athletic Performance in Females

specific actions of performance. Looking at floorball-specific motor tasks, Krasňanová et al. (2014) found

no significant improvement in shooting accuracy, standing long jump, or performance in the 6 x 9m

shuttle run in women supplemented with an average of 4.4mg/kg caffeine. There was a significant

improvement in the average completion time of the longer, 6 x 40m shuttle run, which has been shown

to reliably reflect power and anaerobic capacity (Baker et al. 1993). There was a large standard deviation

in the results of this data, 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 3mg/kg caffeine on the anaerobic performance of female professional

basketball players. Small to moderate significant increases in 10m and 20m sprint times and a moderate

decrease in RPE were reported in the supplemented group, along with small nonsignificant

improvements in squat jump, agility, and suicide run 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.

Commercially-available energy drinks are a marketed source of pre-workout 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 3mg/kg caffeine in the form of a commercial

energy drink 60 minutes prior to exercise. No effect of caffeine was seen on maximal running speed in

sprint testing, but total leg muscle power in 15s maximal jump testing improved, as well as average

running pace during simulated game play, showing some ergogenic benefits of the commercial drinks in

this population of female rugby athletes. Female soccer players who supplemented with 3mg/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
Dietary Supplements for Athletic Performance in Females

significant difference in maximal speed during game play. Astorino et al. (2012) supplemented female

soccer payers with the popular energy drink Red Bull, which contains only 1.3mg/kg caffeine, one hour

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 in

comparison to the positive results of higher-dose studies, suggests that the 1.3mg/kg caffeine provided

in 1 serving of Red BullÒ is an insufficient dose to be effective in female athletes. Given the popularity of

energy drinks, it is reasonable to assume that non-athletes also consume these substances with the

intention of reaping their advertised benefit. In a study of untrained females given commercial energy

drinks at a dose of 4ml/kg, 45 minutes before aerobic exercise, no significant difference was seen

between caffeine and placebo groups for TTE, HR, blood lactate, or VO2max (Al-Fares et al. 2015). It is

important to note that the dosage of energy drink was measured in ml of drink per kg 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 to others mentioned.

Also investigating commercially-available products, Tinsley et al. (2017) compared the effects of

both caffeinated and non-caffeinated commercial energy drink on muscular force production in women

and men. The caffeinated energy drink contained 3.6mg/kg caffeine, and the rest of the ingredients

varied between the two drinks, save for a consistent 6g citrulline malate in both beverages. The

increases in strength seen in the results of a 3-rep max force production test in both men and women

were statistically non-significant. However, the differences between men and women in the

improvements that did occur were much more pronounced in the non-caffeinated group (0-1.5%

increase for women, 9% for men) compared to the caffeinated group (5-11% increase for both women

and men). Skinner et al. (2019) examined the sex-based differences in 3mg/kg caffeine supplementation

on endurance cycling performance and found a significant increase in cycling time trial performance

with caffeine, but no significant difference between sexes. Notably, post-exercise plasma concentrations
Dietary Supplements for Athletic Performance in Females

of caffeine were significantly higher in women than in men, with only women experiencing significant

pre- to post-exercise increases in plasma caffeine concentration. While both of these 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 to non-caffeinated

energy drinks may support the idea of different metabolic responses to caffeine in women compared to

men. This possibility is exemplified by Potgieter et al. (2018), who found a greater magnitude of

response to 6mg/kg caffeine in triathlon performance in men compared to women.

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 (DOMS) and exercise-induced muscle

damage (EIMD) in both male and female college athletes. One hour after ingestion of 6mg/kg caffeine,

men saw a significantly greater decrease in reported DOMS compared to women. This study also

showed a reverse correlation between the reduction in DOMS and the restoration of power output after

EIMD in men, but no such correlation was found in women. While this suggests that caffeine has an

ergogenic effect on perceived muscular pain post-exercise only in men, Motl et al. (2006) found a

significant reduction in mid-workout pain perception in moderately-trained college women given either

5mg/kg or 10mg/kg caffeine one hour before exercise. Decreased perception of pain mid-workout could

translate to longer or harder training sessions in women, leading to greater physiological adaptation to

training. The similar efficacy of the 5mg/kg and 10mg/kg doses suggests that there may be a threshold

after which the dose-response effect of caffeine plateaus.

Nitrate

As made clear in the review by Wickham et al. (2019), the sex-based disparity in research on dietary

nitrate supplementation is wide. 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.
Dietary Supplements for Athletic Performance in Females

Investigating aerobic effects of nitrate on an exercise time trial performance, Peeling et al.

(2015) saw a significant 1.7% increase in 50m time trial performance in elite female kayak athletes after

an acute dose of ~9.6mmol of dietary nitrate in the form of beetroot juice 2 hours prior to exercise. This

study, however, did not measure plasma nitrate or NO2- to verify that positive changes in performance

could be due to the effects of nitrate pathways. Pospieszna et al. (2016) found significant improvement

in swimming time trials in female university-level swimmers after chronic 8-day beetroot juice

supplementation with only 5.1mmol of dietary nitrate per day.

Three additional time trial studies saw no significant effect of dietary nitrate on performance. In

recreationally active females supplemented both acutely (2 hours before exercise) and chronically (8

days) with ~26mmol nitrate per day, there was no significant improvement in cycling time trial

performance or 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 ~7.3mmol

nitrate 2.5 hours before exercise and 5mg/kg caffeine 1 hour before exercise, time trial performance

was not significantly altered by NO3-, regardless of caffeine status (Glaister et al. 2015). A similar study in

elite Olympic-level cyclists showed no effect on time trial performance with two 8.4mmol doses of

nitrate both 10-12 hours and ~2 hours prior to exercise. In those athletes also supplemented with

3mg/kg caffeine, there were no additive benefits of nitrate seen compared to 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 from beetroot juice supplementation investigated swimming and kayaking, both upper-

body heavy exercises. This may suggest that dietary nitrate is more efficacious in upper-body compared

to lower-body endurance sports.

It has also been suggested that dietary nitrate supplementation has greater efficacy in untrained

and moderately trained individuals compared to highly trained athletes or exercisers (Senefeld et al.
Dietary Supplements for Athletic Performance in Females

2020; Hoon et al. 2013). While reviews which have suggested higher efficacy with less training have

done so using primarily male data, the only mentionable study in untrained women, Fernandes de

Castro et al. (2018) found no significant improvement in 3km race performance with acute pre-race

supplementation with 8.4mmol NO3-. Despite the contradiction presented by this one study in women,

replication of such 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 12.9mmol nitrate, 150 minutes prior to 30 minutes of exercise on a cycle

ergometer, the middle 20 minutes occurring at an RPE of 13. Dietary nitrate did not have an effect on

total VO2 or rate of mechanical work compared to 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 significant change in the performance outcomes of this study

suggests that supplementation with dietary nitrate may not have any notable benefits to the quality of

daily training in female athletes, especially when exercise 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, despite an increase in plasma NO3-, in elite female water polo players

after 6 days of chronic beetroot juice supplementation totaling ~12mmol nitrate per day. Similarly, the

effects of 6mmol nitrate supplemented 3 hours 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. On the other hand, the
Dietary Supplements for Athletic Performance in Females

study by Pospieszna et al. (2016) which found improvements in swimming time trial performance with 8

day chronic supplementation of 5.1mmol nitrate also found changes in anaerobic sprint swimming

performance of even greater significance. While this outcome could suggest that dietary nitrate

provides more pronounced ergogenic benefits in anaerobic activity compared to 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 not only the effect of nitrate supplementation on the

performance on amateur runners, but also the extent to which sex may play a role in such effects. 10

women and 14 men were given ~12.8mmol nitrate 2.5 hours before executing a 2km time trial. There

was a significant improvement in time trial performance and a reduction in general RPE, however the

magnitude of the response to supplementation 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 frequency of use in recreational, amateur competitive, and professional athletes,

beta-alanine, caffeine, and nitrate, are three 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 leading to longer exercise sessions and allowing for 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
Dietary Supplements for Athletic Performance in Females

status, with some individuals reacting more strongly to the supplement 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 studies which do exist are not without limitations (addressed

in the following section), which impacts their applicability.

Limitations of Current Literature

Hormonal Status

Of the 34 studies addressed in this review, all of which included female participants, only 10

controlled for menstrual cycle and hormonal status in some way. Menstrual cycle phase in women and

hormonal status have been shown to impact exercise metabolism (Tarnopolsky 2008; Devries et al.

2006), beta-alanine metabolism (Draper et al. 2018), caffeine clearance rates (Lane et al. 1992;

Patwardhan et al. 1980), and plasma NO3-: NO2- ratios (Chatterjee and Mukhopadhyay 2015). Of the 10

studies which took the cyclic fluctuations of sex hormones into account, 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 in regard to exercise physiology and nutritional supplementation (Elliott-Sale et al. 2021).

However, by ignoring one of the major physiological reasons why “women are not small men,” this

research limits itself in generalizability to the female population.

Race
Dietary Supplements for Athletic Performance in Females

Racial differences have been identified in the metabolism of micronutrients and their

relationships to other metabolic biomarkers (Gutiérrez et al. 2011; Suarez and Schramm-Sapyta 2014). It

is feasible that race and ethnicity are also associated with an individuals’ metabolism and utilization of

ergogenic dietary supplements such as beta-alanine, caffeine, and nitrate. There is a stark lack of regard

for race in the studies outlined in this review. Only 3 of 34 studies presented data on race, and 2 of

these 3 included Caucasian participants only. A total of 652 individuals participated in the 34 studies: of

these, there is explicit mention of only 6 Hispanic, 3 Asian, and 1 African American participant. As

research on ergogenic aids and female athletes progresses, it is important to ensure inclusion of the

diverse ethnic population that is represented in our nation.

Age

The majority of these studies (30/34) were conducted in women of mean age below 30 years.

While athletic performance generally begins to decline after age 30, many individuals continue to

exercise both recreationally and competitively past well past the conclusion of their “peak performance”

years of their 20s (Ganse et al. 2018). Hormonal status also changes as women age, with declines in

estrogen and progesterone beginning as early as the 30s (Hale and Burger 2009). By not commonly

including women past the age of 30, researchers could be missing other important relationships

between women and performance enhancing supplements that exist by way of hormonal interactions

or lack thereof. For this reason, it may be prudent to include women of a greater range of ages in future

studies of dietary supplements and exercise. Moreover, the understanding that physical activity even

later in life continues to promote longevity and reduce disease risks makes studying exercise and

potentially exercise-enhancing supplements in older women all the more relevant for public health (Mok

et al. 2019).

Training Status
Dietary Supplements for Athletic Performance in Females

The physical fitness and training status of the women participating in these studies varied from

untrained, sedentary to international-level 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 addressed in this review. However, studies in men have suggested

relationships between training status and response to ergogenic aids, specifically nitrate (Senefeld et al.

2020; Hoon et al. 2013). Though no patterns of effect magnitude and training status in women have yet

emerged in regard to dietary nitrate, nor beta-alanine or caffeine, expanding upon the limited depth and

breadth of literature may elucidate relationships between training status and efficacy in the future.

CONCLUSION

Despite the growing awareness of the need for female-specific exercise and nutrition research,

the body of literature on common ergogenic aids, such as 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, continue

to limit the ability to apply outcomes to generalized guidelines for women in sport. 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 commonly-used ergogenic aids and discuss outcomes and

implications specifically regarding women. Further research in women is necessary to continue to

elucidate potential sex-specific dosage, timing, and general efficacy guidelines for beta-alanine, caffeine,

and nitrate. These three supplements continue to be used by athletes and are recognized by professional

organizations as effective ergogenic aids which enhances their likely continued use in the future.
Dietary Supplements for Athletic Performance in Females

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 Table 1 Effects of dietary beta-alanine, caffeine, and nitrate supplementation on exercise performance

Reference Population Number, Age, and Supplement Exercise Protocol Main outcomes Power
Sex of Participants Dosage Analysis
Beta-alanine
Glenn et al. Trained, competitively N = 12 F 1.6g 3 x Wingate trials with 2 mins ↑ RPE
(2015) active cyclists Age = 26.6 ± .3 active rest between ↔ mean power, peak power,
(Avg 3.92 yrs training) HR, BLa
All participants in luteal
phase of menstrual cycle
(48 hrs post-cessation of
menses)
Kresta et al. Moderately Active (≥ 30 N = 32 F 0.1g/kg days GXT (cycle ergometer) and 2 x 30- ↔ mean power, peak power,
(2014) mins 3x/week) adults Age = 21.5 ± 2.8 1-28 second Wingate tests at work rate total work
of 7.5J/kg/rev with 3 mins rest ↑ rate of fatigue
between

Outlaw et al. Untrained adults N = 16 F 3.4g before 4x Bruce Protocol (treadmill), ↔ peak power, max strength,
(2016) Age = 21 ± 2.2 weekly Wingate test, 1RM bench press, bench press reps to failure, VO2,
training 65%1RM leg press TTE
to failure, vertical jump and ↑ Vertical Jump, Standing Broad
standing broad jump tests Jump, Leg press RTF

Ribeiro et al. International level elite N = 24 F 1.6g 4x/day YoYo Intermittent Recovery Test ↔ repeated sprint ability, 20m
(2020) soccer athletes Age = 18 ± 1 (6.4g total) Level 1 (YoYo IR1), sprint time, YoYo IR1
Running Anaerobic Sprint Test
(RAST), and 20m maximal sprint
test
Rosas et al. Amateur soccer athletes N = 25 F 0.8g 6x/day Squat jump, countermovement ↑ 60s countermovement jump 95% power,
(2017) Age = 23.7 ± 2.4 (4.8g total) jump, 20 m sprint test, power, repeated sprint ability, alpha = 0.01
running anaerobic sprint test shuttle run
[RAST], 40 cm drop jump reactive ↔ change of direction speed,
strength index, squat jump, peak jump power,
peak jump power, change-of- reactive strength
direction speed [Illinois test],
20 m multistage shuttle run, and
60 s countermovement jump
Smith et al. Moderately trained (3- N = 24 F 1.6g 3x/day GXT (treadmill) and 40-min ↔ VO2max, VT, TTE, HR
(2012) 7x/week) adults Age = 21.7 ± 2.1 days 1-28 treadmill run at 70% peak velocity ↑ RPE
(4.8g total)
Smith-Ryan et al. Recreationally Active (1- N = 24 F, 26 M 1.6g 3x/day 3 x run to exhaustion at 100%PV, ↔ TTE, critical velocity,
(2012) 5 hrs per week) adults Age = 21.7 ± 2.1 (F), (4.8g total) 90%PV, and 110%PV with 15 anaerobic running capacity
22.0 ± 3.3 (M) minutes of rest between each
bout
Stout et al. Not explicitly mentioned N = 22 F 3.2g/day days GXT (cycle ergometer) ↑ VT, working capacity, TTE
(2007) - assumption is either Age = 27.4 ± 6.1 1-7 ↔ VO2max
untrained or 6.4g/day days
recreationally active 8-28
adults
Walter et al. Recreationally Active (1- N = 44 F 1.5g BA GXT (cycle ergometer) weeks 0, 4, ↔ Vo2peak, Power at VT
(2010) 5 hrs per week) adults Age = 21.8 ± 3.7 4x/day weeks and 8.
1-4 (6g total) 30 mins of HIIT on the bike 3x per
1.5g BA week, weeks 1-3 and 5-7
2x/day week
5-7 (3g total)
Caffeine
Al-Fares et al. Untrained (not regularly N = 32 F 4ml/kg BW Bruce Protocol (treadmill) ↔ TTE, Vo2max, HR, BLa 90% power,
(2015) exercising) adults Age = 19.9 ± 0.8 ↑ BP alpha = 0.05
Ali et al. Varied: recreationally N = 10 F 6mg/kg BW 2 x 15 min blocks of intermittent ↑ knee flexor eccentric toque
(2016) active adults --> Age = 24 ± 4 treadmill running protocol ↔ knee flexor concentric
internationally Eccentric, concentric, and torque, knee extensor eccentric
competitive athletes isometric contractions of knee + concentric torque, isometric
All on oral flexors and strength, jump height, power,
contraceptives, with extensors (isokinetic Vo2, HR
exercise testing dynamometer)
occurring on same days
of cycle
Astorino et al. College soccer athletes N = 15 F 1.3mg/kg BW 3 x 8 “all-out” t-test sprints w/ 5 ↔ sprint performance, RPE, HR
(2012) (~12 hr training/week) Age = 19.5 ± 1.1 mins rest between sets

Chen et al. Division 1 college N = 10 F, 10 M 6mg/kg BW Downhill running protocol to ↑ muscle performance M and F
(2019) athletes Age = 20.4 ± 1.2 (F), induced DOMS/EIMD. ↑ DOMS, M>F
No oral contraceptive 21.1 ± 2.1 (M) 24 and 48hrs after EIMD, muscle ↑ EIMD M only
use, all exercise testing performance measured using
in early follicular phase knee flexor/extensor peak power
of menstrual cycle on a dynamometer
Del coso et al. National-level rugby N = 16 F 3mg/kg BW 6 x 30m sprint test, 3 rugby ↔ max running speed, HRmax
(2013) "sevens" athletes Age = 23 ± 2.0 competition games with 15 mins ↑ leg muscle power, mean
rest between running pace
games, 15-s jump test
Goldstein et al. Resistance trained (3-5 N = 15 F 6mg/kg BW 1RM barbell bench press test and ↑ 1RM
(2010) days/week) Age = 24.6 ± 6.9 RTF at 60% of 1RM ↔ RTF, HR, BP

Krasňanová et al. Competitive floorball N = 14 F 4.4mg/kg BW Standing long jump, 6 x 9m ↑ 6x40m shuttle run time
(2014) team athletes Age = 23.3 ± 5.3 shuttle run, reaction speed test, ↔ 9 x 40m shuttle run time, leg
shooting accuracy test, and 6 x muscle power, shooting
40m shuttle run accuracy, reaction speed

Lara et al. Competitive soccer N = 18 F 3mg/kg BW Jump test, 7 x 30m max running ↔ max sprint speed
(2014) athletes (6-8 hrs Age = 21 ± 2.0 speed test, and a simulated ↑ jump height, peak power,
training/week) soccer game (2 x 40min halves) peak sprint speed, # and length
of sprints
Motl et al. Adults of average fitness N = 11 F 5mg/kg BW 30 mins cycling at 60% VO2peak ↔ power, HR, SBP, VO2 80% power,
(2006) (not sedentary but not Age = 19.5 ± 1.1 10mg/kg BW ↑ pain intensity ratings alpha = 0.05
trained)
No oral contraceptive
use, all exercise testing
occurred in self-
reported follicular phase
of menstrual cycle
Norum et al. Resistance trained (2-3 N = 15 F 4mg/kg BW 1RM, RTF at 60% of 1RM, ↑ 1RM, RTF, MVC torque, 80% power,
(2020) days/week) adults, all Age = 29.8 ± 5.5 isometric knee extensions, countermovement jump, power alpha = 0.05
subjects Caucasian countermovement jump ↔ force, muscle activation
All exercise testing
occurred in early
follicular phase of
menstrual cycle,
some participants using
oral contraceptives and
some not

Perez-Lopez et al. National-level elite N = 13 F 3mg/kg BW standing spike and jump spike, ↑ handgrip, max ball spike
(2015) volleyball athletes Age = 25.2 ± 4.8 maximal spike jump, velocity, jump height, agility T-
Exercise testing squat/countermovement/block test, HRmean, HRmax
occurred in follicular jumps, max manual dynamometry
phase (n = 4) and luteal (hand grip), and agility t-test (all
phase (n = 9) x2 with 1 min
between reps and 3 mins
between tests)
Potgieter et al. Competitive triathletes, N = 12 F, 14 M 6mg/kg BW Triathlon ↑ overall time M only, swim
(2018) all subjects Caucasian Age = 37.2 ± 11.6 (F), time
38.4 ± 10.0 (M) ↔ RPE

Skinner et al. Competitive N = 11 F, 16 M 3mg/kg BW 2 x cycling TT ↔ RPE 80% power,

(2019) cyclists/triathletes Age = 29.7 ± 5.3 (F), ↑ cycling performance, HR alpha = 0.05
All participants using 32.6 ± 8.3 (M)
oral contraceptives, all
exercise testing
 occurred
in luteal phase of
menstrual cycle

Stojanovic et al. Professional basketball N = 10 F 3mg/kg BW Jump test with and without arm ↑ 10m and 20m sprint times 80% power,
(2019) athletes Age = 20.2 ± 3.9 swing, squat jump, ↔ jump height, repeated sprint alpha = 0.05
lane agility drill, 20m sprints with performance, lane agility drill
and without dribbling, and 140m
suicide run
Tinsley et al. Resistance trained (7.5 ± N = 12 F, 9 M 3.6mg/kg BW 3-rep max force production test ↔ concentric force, 80% power,
(2017) 3.9 hr/week) adults Age = 21.5 ± 2.0 (F), and 5 x 6 isokinetic squats at eccentric force, RPE, fatigue alpha = 0.05
Subjects included 11 20.7 ± 2.8 (M) perceived
Caucasian, 6 Hispanic, 3 maximal effort
Asian, 1 African
American
Nitrate
Buck et al. Amateur team-sport N = 13 F 6mmol Simulated team-game circuit ↔ sprint times, RPE, HR
(2015) athletes (195 ± 42 min Age = 25.5 ± 1.9 (STGC) consisting of four 15 min
play/week) quarters, 3 x RSA
n = 9 participants using
oral contraceptives, n =
4 participants not using
oral contraceptives
Casado et al. Amateur runners N = 10 F, 14 M 12.8mmol 2 km running TT ↑ RPE M and F
(2021) Age = 36.6 ± 8.2 (F), ↑ TT M and F
38.7 ± 9.2 (M) ↔ BLa

Fernandes de Untrained adults N=8F 8.4mmol 2 x 3 km running TT ↔ TT performance, HRmax, Bla

Castro et al. Age = 30 ± 5.7 ↑ RPE
(2018)

Glaister et al. Competitive N = 14 F ~7.3mmol 4 x 20 km cycling TT ↔ VO2, RPE, HR, BLa

(2015) cyclists/triathletes (10.7 Age = 31 ± 7
± 2.2 hrs training/week)
Jovnik et al. National-level Water N = 14 F ~12mmol Intermittent swim sprint test 4x4 ↔ swim sprint performance,
(2018) Polo athletes Age = 22 ± 4 15m sprints w/ 30s active rest dynamic apnea distance
between sets
Lane et al. International-level N = 12 F, 12 M 8.4mmol x 2 Cycling TT (29.35 km F, 43.83 km ↔ TT performance, mean
(2014) competitive Age = 28 ± 6 (F), (16.8 mmol M) power
cyclists/triathletes 31 ± 7 (M) total)
Peeling et al. International (Olympic) N=5F ~9.6mmol 50m Kayak Paddle TT ↑ TT performance
(2015) level kayak athletes Age = 25 ± 2.8

Pospieszna et al. University league N = 11 F 5.1mmol/day 6 x 50 m maximal swim sprint, ↑swim sprint performance,
(2016) swimmers (3 Age = 20.9 ± 1.3 x 8 days 800m continuous swim endurance TT
practices/week) ↔ HR, BP

Rienks et al. Recreationally active N = 10 F 12.9mmol "RPE Clamp" Protocol at RPE of 13 ↑ VO2 at fixed work rate
(2015) (Vo2 peak ~35 Age = 25 ± 3 ↔ total VO2, total work
ml/kg/min) adults
Wickham et al. Recreationally active Study 1: O2 and ~26mmol Study 1: submaximal cycling ↔ TT performance, VO2
(2019) (Vo2 peak 35-35 performance nitrate x 1 day protocol, 4 kJ/kg BW cycling TT ↑ plantar flexor torque
ml/kg/min, 4.5 ± 1.5 h N = 12 F and x 8 days Study 2: 3 x MVC of plantar
exercise/week) adults Age = 23 ± 1 flexors with 3 mins rest between
All participants using Study 2: contractile tests, Electric current stimulation
hormonal properties to achieve 30% MVC at 100 Hz
contraceptives N = 12 F (N=7 from
Study 1)
Age = 22 ± 2
Abbreviations: ↑, improvement; ↔, no change; RPE, rate of perceive exertion; HR, heart rate; BLa, blood lactate; BW, body weight; 1RM, one-repetition
maximum; RTF, repetitions to failure; GXT, graded exercise test; TTE, time to exhaustion; PV, peak velocity; VT, ventilatory threshold; TT, time trial; MVC,
maximum voluntary contraction; (S)BP, (systolic) blood pressure; VO2, oxygen consumption; DOMS, delayed-onset muscle soreness; EIMD, exercise-
induced muscle damage.