Abstract
kground: Inorganic nitrate (NO3 −) may enhance muscle contractility and blood flow via nitric oxide production, offering potential ergogenic benefits. However, most studies have focused on males, and its effects in females during high-intensity, short-duration exercise remain unclear. Objective: This review assessed the acute effects of nitrate supple- mentation on muscle strength, power, and sprint performance in healthy females. Methods: A comprehensive literature search was conducted in PubMed, Web of Science, Scopus, SPORTDiscus, and Cochrane CENTRAL, from inception to July 2025. Randomized con- trolled trials (RCTs) that examined the effects of oral inorganic nitrate (e.g., beetroot juice or nitrate salts) in healthy females were included. Eligible studies involved (i) healthy women aged 18–30, (ii) nitrate supplementation vs. placebo, and (iii) outcome measures of muscle strength(e.g., MVC),power (e.g., countermovement jump height, peak power), or sprint performance (e.g., sprint time or repeated sprint ability). Studies were excluded if they did not report sex-specific results or lacked relevant physical performance outcomes. Random- effects meta-analyses were conducted for each outcome. Results:
involved (i) healthy women aged 18–30, (ii) nitrate supplementation vs. placebo, and (iii) outcome measures of muscle strength(e.g., MVC),power (e.g., countermovement jump height, peak power), or sprint performance (e.g., sprint time or repeated sprint ability). Studies were excluded if they did not report sex-specific results or lacked relevant physical performance outcomes. Random- effects meta-analyses were conducted for each outcome. Results: Nitrate supplementation had small, non-significant effects on muscle strength (SMD = 0.10,95% CI:−0.10 to 0.30, p> 0.05 ) and sprint performance (SMD = 0.14, 95% CI:−0.13 to 0.41,p> 0.05). A statis- tically significant, small-to-moderate improvement was observed in power (SMD = 0.38, 95% CI: 0.06 to 0.69,p< 0.05). Sensitivity analyses confirmed robustness of the power result. The certainty of evidence ranged from low to moderate based on the GRADE assessment. Conclusions: Inorganic nitrate supplementation may modestly enhance power in healthy females but does not appear to significantly improve muscle strength or sprint performance. These findings highlight the importance of sex-specific research in sports nutrition to guide targeted supplementation strategies for female athletes. Keywords:inorganic nitrate; beetroot juice; power; muscle strength; sprint performance; female athletes 1. Introduction In recent years, nutritional supplements have gained increasing attention as strategies to optimize athletic performance. Beyond training adaptations, specific nutrients may Life2025,15, 1425 https://doi.org/10.3390/life15091425
Life2025,15, 1425 2 of 18 enhance energy metabolism, muscle function, and fatigue resistance, thereby offering performance benefits that exceed those achieved by training alone [1,2]. Among the most extensively studied supplements, inorganic nitrate (NO3 −) has emerged as a promising ergogenic aid due to its involvement in the nitric oxide (NO) pathway, which regulates blood flow and metabolism [3,4]. NO3 −is naturally abundant in green leafy and root vegetables, including beetroot, spinach, lettuce, and celery [5]. Beetroot juice (BRJ), with its high nitrate content, has become the most common form of supplementation and is widely used in pre-training and pre- competition nutritional strategies [5]. Following ingestion, dietary nitrate enters the entero- salivary-gastric circulation, where it is reduced to nitrite (NO2 −) by anaerobic bacteria in the oral cavity, and subsequently converted to NO under hypoxic or acidic conditions [6]. This NO3 −–NO2 −–NO pathway functions independently of the nitric oxide synthase (NOS) and may be particularly relevant during high-intensity or oxygen-limited exercise [7]. As a key signaling molecule [8], NO plays a crucial role in vasodilation [9], skeletal muscle blood flow [10], mitochondrial efficiency, and excitation–contraction coupling, thereby supporting oxygen delivery and energy supply, muscle performance [11,12]. Nevertheless, findings regarding their effects on muscle strength and power remain inconsistent, suggesting the need for further targeted investigations [13]. Early research on nitrate supplementation, particularly BRJ, focused on aerobic en- durance exercise at moderate to submaximal intensities, such as cycling [14–17] and dis- tance running [18–22]. While individual studies produced mixed results, several systematic reviews and meta-analyses have consistently demonstrated that nitrate supplementation can reduce oxygen consumption and energy expenditure, improve exercise economy, and prolong time to exhaustion [23–27]. More recently, attention has shifted to high-intensity, short-duration, neuromuscularly demanding activities [28–33], which rely primarily on the phosphocreatine (PCr) energy system and recruit large numbers of type II fast-twitch mus- cle fibers [34]. For instance, its effects on power, maximal strength, and sprint performance have become focal points in current investigations [33]. Power—the ability to produce maximal mechanical output in minimal time—is critical for performing sport-specific actions including jumping, sprinting, and change in
rely primarily on the phosphocreatine (PCr) energy system and recruit large numbers of type II fast-twitch mus- cle fibers [34]. For instance, its effects on power, maximal strength, and sprint performance have become focal points in current investigations [33]. Power—the ability to produce maximal mechanical output in minimal time—is critical for performing sport-specific actions including jumping, sprinting, and change in direction, and is a key determinant of athletic success [35,36]. While resistance training promotes strength and power through neural and structural adaptations, explosive movements are often limited by PCr depletion and calcium handling impairments [37]. Some studies have reported that nitrate supplementation can attenuate PCr depletion, enhance calcium reuptake, and improve excitation–contraction coupling. These effects are thought to involve both improved mitochondrial efficiency, which reduces the rate of PCr utilization, and enhanced sarcoplasmic reticulum Ca 2+ handling that facilitates muscle contraction and relaxation [11,38]. While increased muscle blood flow may play a role, intramuscular mechanisms appear particularly important in supporting force output and endurance capacity during repeated high-intensity efforts [32,39,40]. Although several trials have demonstrated improvements in peak power output (Pmax), number of repetitions, and short-duration sprint performance following nitrate supplementation, findings remain inconsistent. Some studies have shown significant positive effects [41–43], while others reported no difference or highly variable individual responses [28,40,44]. Variability may be influenced by training status, supplementation dose and timing, or outcome measures. A critical limitation of current literature is the underrepresentation of female partici- pants. Most studies have been conducted in males or mixed cohorts, limiting sex-specific insights [45]. Yet females differ from males in several physiological domains, including hormone profiles, skeletal muscle composition, NO bioavailability, and fatigue recovery mechanisms [46,47]. The anabolic effects of testosterone result in greater muscle mass in
Life2025,15, 1425 3 of 18 males, which not only influences the response to supplementation, but may also affect the storage and utilization efficiency of nitrate and nitrite within the muscle tissue [48]. Conversely, some evidence suggests that females may experience greater increases in plasma NO2 −following nitrate ingestion, potentially translating into performance gains in explosive tasks such as jumping or sprinting [31,49,50]. However, other findings indicate a weaker or even null ergogenic effect in females under certain conditions [51–55]. With the rising participation of women in competitive and strength-based sports, reliance on male-centric data to guide nutritional strategies is increasingly inadequate. Addressing this gap is crucial for both scientific progress and applied practice. Therefore, this systematic review and meta-analysis specifically examines the effects of inorganic nitrate supplementation on muscle strength, power, and sprint performance in female athletes. This study aims to provide robust, sex-specific evidence for practitioners and help guide future research into sex-related responses to nitrate supplementation. 2. Methods This meta-analysis adhered to the PRISMA 2020 guidelines (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) to ensure transparency and completeness in reporting [56]. In line with open science practices and to promote reproducibility, the review protocol was prospectively registered on the PROSPERO database (https://www.crd.york. ac.uk/prospero/ 2.1. Eligibility Criteria The inclusion and exclusion criteria were developed according to the PICOS frame- work (Participanst, Intervention, Comparison, Outcomes, Study design) to ensure that all included studies addressed clearly defined populations, interventions, comparators, outcomes, and methodological rigor [57], as follows: Participanst (P): Eligible studies included healthy female participants, typically aged between 18 and 35 years, regardless of athletic status. Participants could be recreationally active, physically trained, or competitive athletes. Studies that included mixed-sex samples were considered only if sex-specific data for females were reported separately. Animal studies and non-human trials were excluded during the title and abstract screening phase. Intervention (I): Studies were required to evaluate the effects of oral inorganic nitrate supplementation—most commonly in the form of beetroot juice—administered acutely (e.g., single dose 2–3 h pre-exercise). There were no restrictions on dosage or delivery form, but the intervention protocol had to be
reported separately. Animal studies and non-human trials were excluded during the title and abstract screening phase. Intervention (I): Studies were required to evaluate the effects of oral inorganic nitrate supplementation—most commonly in the form of beetroot juice—administered acutely (e.g., single dose 2–3 h pre-exercise). There were no restrictions on dosage or delivery form, but the intervention protocol had to be clearly described, including the amount of nitrate provided and timing relative to performance testing. Comparison (C): Eligible studies had to include a placebo or non-supplemented control group under equivalent experimental or training conditions. In multiarm trials, comparisons between the nitrate group and a clearly defined placebo or control group were required. Outcomes (O): Studies were eligible if they re-ported at least one quantitative outcome related to physical performance, specifically focused on domains such as muscle strength (e.g., maximal voluntary isometric con-traction or isokinetic peak torque), power (e.g., countermovement jump height, rate of torque development, or peak power), or sprint performance (e.g., linear sprint time over 10–30 m or repeated sprint ability). Studies were included regardless of whether outcomes were measured in laboratory or sport-specific field tests, as long as the methodology and performance metrics were clearly described and extractable. Study design (S): Only randomized controlled trials (RCTs) or random- ized crossover trials, published in peer-reviewed English language journals, were eligible for inclusion. Studies were excluded if they met any of the following conditions: (i) No female- specific data were reported in mixed-sex trials; (ii) Use of multi-ingredient supplements
Life2025,15, 1425 4 of 18 without isolating the effect of nitrate; (iii) Absence of a placebo or control group; (iv) Lack of relevant performance outcomes or outcomes not extractable; (v) Non-original studies such as reviews, conference abstracts, theses, or protocols; (vi) Full text or critical data unavailable even after author contact. 2.2. Data Sources and Search Strategy A comprehensive search of the literature was performed using five electronic databases: PubMed, Scopus, Web of Science, Cochrane CENTRAL, and SPORTDiscus, aiming to identify randomized trials that examined the effects of inorganic nitrate supple- mentation on muscle strength, explosive power, or sprint performance in female partici- pants. To enhance the completeness of the search, additional strategies were employed, including manual screening of reference lists from all included articles, forward citation tracking through Google Scholar, and the use of the “Similar Articles” function in MEDLINE and Embase. Only peer-reviewed full-text publications in English were considered eligible. The search was independently conducted by two reviewers (F.M. and J.L.), covering studies published from database inception to 17 July 2025, with no restrictions applied to the year of publication. Boolean phrases and keywords used are detailed in Supplementary Table S2. 2.3. Study Selection and Screening Process All retrieved records were first manually de-duplicated by an independent reviewer (F.M.) using EndNote X9 (Clarivate Analytics, Philadelphia, PA, USA). The resulting unique records were then screened independently by two reviewers (F.M. and J.L.) following the predefined inclusion and exclusion criteria. Title and abstract screening was performed to identify studies for full-text review. Any disagreements during this phase were resolved through discussion, and if needed, a third reviewer (Y.L.) was consulted to reach a consen- sus. Full-text articles were subsequently assessed in the same manner by thetwo reviewers to confirm final eligibility, with the same conflict resolution process applied when neces- sary. In addition, two supplementary approaches were used to identify potentially missed studies: (i) reviewing the reference lists of existing systematic reviews on related topics, and (ii) leveraging the reviewers’ expertise to locate relevant studies not captured in the initial database search. 2.4. Data Extraction and Transformation Two reviewers (F.M.
eligibility, with the same conflict resolution process applied when neces- sary. In addition, two supplementary approaches were used to identify potentially missed studies: (i) reviewing the reference lists of existing systematic reviews on related topics, and (ii) leveraging the reviewers’ expertise to locate relevant studies not captured in the initial database search. 2.4. Data Extraction and Transformation Two reviewers (F.M. and J.L.) independently extracted data from each eligible study, and a third reviewer (B.Q) verified the accuracy and completeness of the extracted informa- tion. All data were compiled and managed in Microsoft Excel ® (Microsoft Corporation, Redmond, WA, USA) using a standardized extraction template. The following variables were collected: (i) Study characteristics: first author, year of publication, country, sample size, participant sex (female-specific), age, training status, and sport discipline; (ii) Inter- vention details: form and dosage of inorganic nitrate (e.g., beetroot juice), duration and timing of supplementation and control condition; (iii) Performance outcomes: measures of muscle strength (e.g., maximal voluntary isometric contraction, isokinetic peak torque), power (e.g., countermovement jump height, peak power, rate of torque development), and sprint performance (e.g., 10–30 m sprint time, repeated sprint performance); (iv) Study design and other relevant information: study type (randomized controlled or crossover trial), performance test timing (pre- and post-intervention), and any reported adverse effects. When outcome data were only available in graphical format, numerical values were estimated using WebPlotDigitizer (version 4.1; (accessed on 21 July 2025)) [58].
Life2025,15, 1425 5 of 18 2.5. Risk of Bias and Certainty Assessment The risk of bias for each included study was assessed using the Cochrane Risk of Bias 2.0 (RoB 2.0) tool [59], which evaluates five core domains: (i) The randomization process, (ii) Adherence to intended interventions, (iii) Completeness of outcome data, (iv) Measurementof outcomes, and (v) Selection of reported results. Two reviewers (F.M. and J.L.) independently performed the assessments, and any disagreements were resolved through discussion with a third reviewer (B.Q.) to reach consensus. A traffic light plot and weighted summary figure of the risk of bias across included studies were generated using the robvis visualization tool (https://mcguinlu.shinyapps.io/robvis/ 24 July 2025)) [60]. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to assess the certainty of evidence across outcomes, with ratings catego- rized as high, moderate, low, or very low [61]. The GRADE assessment was conducted by one reviewer (F.M.) and independently verified by a second reviewer (Y.L.). 2.6. Statistical Analysis All meta-analyses were performed using a random-effects model to accommodate heterogeneity across studies. Effect sizes were expressed as standardized mean differ- ences (SMD), specifically calculated using the Standardized Mean Change with Correlation (SMCC) method, which is suitable for crossover or paired pre–post designs. This approach accounts for within-subject correlations, and is considered more precise than traditional change score methods for repeated-measures data [62], with a default correlation coeffi- cient of r = 0.7, consistent with commonly recommended values for repeated measures outcomes [63,64]. A sensitivity analysis was conducted to assess the impact of varying r assumptions (range: 0.5–0.9) on the robustness of the pooled results. When data were available only in graphical format, numerical values were extracted using WebPlotDigitizer (version 4.1). For studies that reported standard errors instead of standard deviations,SDwas estimated using the formula: SD=SE× √ n (1) Due to variations in measurement units or test protocols across studies, SMD was consistently adopted as the summary effect size. To improve the consistency of outcome cat- egorization across studies, countermovement jump (CMJ) and peak power were combined and analyzed together as a single construct representing
standard errors instead of standard deviations,SDwas estimated using the formula: SD=SE× √ n (1) Due to variations in measurement units or test protocols across studies, SMD was consistently adopted as the summary effect size. To improve the consistency of outcome cat- egorization across studies, countermovement jump (CMJ) and peak power were combined and analyzed together as a single construct representing lower-limb power. This decision was based on their similar physiological basis (i.e., reliance on rate of force development and neuromuscular output), and their common usage in the literature as interchangeable or complementary markers of power [35]. Effect sizes were interpreted using conventional thresholds, with 0.2 considered small, 0.5 moderate, and 0.8 large, as recommended in previous guidelines [65]. Heterogeneity was assessed using theI 2 statistic, with the following interpretation: 0–40% (might not be important), 30–60% (moderate), 50–90% (substantial), and 75–100% (considerable heterogeneity). Additionally, the Jackson method was used to compute tau 2 (τ 2 ), tau (τ), and their 95% confidence intervals (CIs) [66]. Due to the limited number of studies per outcome (n≤10), publication bias was not formally assessed using funnel plots or Egger’s regression test [57]. All analyses and visualizations were conducted using R (version 4.2.0) with the meta and metafor packages. Statistical significance was set atp< 0.05, while values between 0.05 and 0.10 were interpreted as suggestive of a trend.
Life2025,15, 1425 6 of 18 3. Results 3.1. Literature Search A total of 1041 records were initially retrieved through database searches, including PubMed (n= 199), Web of Science (n= 139), Scopus (n= 117), SPORTDiscus (n= 475), and Cochrane CENTRAL (n= 111). After removing 311 duplicate entries, 730 unique records remained for title and abstract screening. Based on predefined eligibility criteria, 705 records were excluded at this stage due to irrelevance to the topic. Subsequently, 25 full-text articles were retrieved for further assessment. After full- text evaluation, 16 studies were excluded—specifically, 4 due to inappropriate population characteristics, 11 for lacking relevant outcome data, and 1 due to the unavailability of full text (we attempted to contact the author without success, and the study did not appear to be peer reviewed). Ultimately, 9 randomized controlled trials (RCTs) met all inclusion criteria and were incorporated into the final analysis. The detailed study selection process is depicted in the PRISMA flow diagram (Figure). Figure 1.PRISMA Flow Diagram of Study Selection. 3.2. Study Characteristics 3.2.1. Participants This systematic review included nine randomized controlled trials involving a total of 114 healthy female participants aged between 18 and 30 years (Table). The participants ranged from recreationally active women to trained athletes, including team-sport players, semi-professional rugby athletes, and elite performers in sports such as field hockey and water polo [49,52,67]. Two studies included both sexes but reported sex-specific outcomes, and was thus eligible [54,68]. All participants were free from chronic diseases and reported no medical contraindications to physical activity or supplementation.
Life2025,15, 1425 7 of 18 Table 1.Characteristics of the Studies Included in the Systematic Review and Meta-Analysis of the Acute Effects of Beetroot Juice Supplementation in Women. Study Country Population Description BRJ Dose and Schedule PL Dose and Schedule Supplement Taking Time Exercise Type Training Details Outcomes Measured Tool Main Outcomes Jurado-Castro (2022) [31] Spain 14 Physically active women (Age: 25.36±3.97 years) 70 mL Beetroot juice (400 mg nitrate) 70 mL blackcurrant juice (nitrate-depleted juice) 2 h pre-exercise Resistance Training 3 sets×3 exercises (75% 1RM), squat/leg press/leg ext. to failure MyJump2, Speed4Lift, 75% 1RM test, RPE CMJ, endurance reps, RPE Tan (2024) [51] USA 15 Trained team-sport athletes (Age: 20±1 years) Beetroot Juice (12.0 mmol NO3 − ) Beetroot Juice (0.1 mmol NO3 − ) 2.5 h before test Mixed: Sprint, Jump, Yo-Yo Test NR Timing gates, jump mat, handgrip dynamometer, Borg scale, Stroop test Sprint (10 m, 20 m), CMJ, Handgrip, Ball throw, RPE, Cognitive flexibility, Oral microbiota López-Samanes a (2023) [52] Spain 11 Elite female hockey athletes (Age: 18.0±1.0 years) 70 mL Beetroot Juice (6.4 mmol NO3 − ) 70 mL Beetroot Juice (0.04 mmol NO3 − ) 2.5 h before test Field hockey Match simulation: 2×12.5 min; 1-week washout between trials Jump mat, handgrip dynamometer, sprint test, GPS tracker CMJ, Handgrip, 20 m sprint, Repeated Sprint Ability, Match GPS data López-Samanes b (2022) [49] Spain 14 Semi-professional rugby women (Age: 25.0±3.7 years) Beetroot Juice (140 mL, 12.8 mmol NO3 − ) Beetroot Juice (Placebo, 0.08 mmol) 2.5 h before testing Rugby Field test Within-subject, 2 visits Jump mat, handgrip dynamometer, sprint test, GPS tracker CMJ, isometric grip, 10/30 m sprint, agility test, Bronco test, RPE, side effects Wickham (2019) [50] USA 12 Recreationally active females using hormonal contraceptives (Age: 21.8±3.4 years) Beetroot juice (280 mL/day, ~26 mmol NO3 − ) nitrate-free placebo 2.5 h before testing cycling, plantar flexor torque Cycling test Plantar flexor torque MVC, voluntary activation, low-frequency torque, RPE, HR Jonvik (2018) [67] Netherlands 14 Elite female athlete (Age: 22±4 years) 4.8 g/day, divided into 3 doses Beetroot juice (140 mL/day, ~800 mg nitrate) Placebo
Description
This systematic review assesses the effects of nitrate supplementation on performance in female athletes.