Acute effects of combined supplementation of L-arginine and citrulline malate on aerobic, anaerobic, and CrossFit exercise performance

Abstract

This study investigated the effects of combined L-arginine (Arg) and citrulline-malate (CM) supplementation on aerobic, anaerobic, and high-intensity interval training in healthy, trained men. Both Arg and CM are widely marketed for their potential ergogenic effects, as Arg serves as a precursor to nitric oxide (NO), which may support vasodilation, muscle contractility, and exercise performance. Arg and CM are hypothesized to exert synergistic effects due to their complementary roles in NO synthesis. Citrulline can potentially enhance and prolong Arg availability, thereby amplifying NO-mediated vasodilation, nutrient delivery, and muscle performance during exercise. In this randomized, double-blind, placebo-controlled trial, 46 healthy, trained men aged 24.8 ± 5.0 years were divided into 3 exercise groups subjected to consuming 0.15 g/kg bodyweight of Arg and 0.1 g/kg bodyweight of CM prior. The participants were then randomly divided into three groups based on the exercise protocol:: the Wingate Anaerobic Test (n = 16), a 20 min CrossFit workout ‘Cindy’ (n = 16) consisting of continuous rounds of pull-ups, push-ups, and air squats to measure functional fitness and muscular endurance, or the Harvard Step Test (n = 14) a measurement of cardiovascular endurance and recovery. This design allowed for the evaluation of supplementation effects across multiple exercise modalities. The results revealed no significant improvement in performance with supplementation in comparison to placebo, except for a shorter time to reach peak power in the Wingate test. Findings suggest that the combined acute supplementation of Arg and CM, at the given dosages, may not provide substantial benefits for aerobic and anaerobic or CrossFit performance in active individuals. Future research with larger sample sizes and higher dosages, potentially adjusted for muscle mass, is recommended to determine whether chronic supplementation might yield greater ergogenic effects.

Introduction

Background

Many nutritional products have been advertised as enhancing the performance, function, and health of athletes. These products, generally categorized as “Sports Foods” or “Dietary Supplements,” are consumed by athletes to improve immune function, training adaptation, recovery, and general health in addition to supporting performance during training and competition¹. According to the International Olympic Committee statement a dietary supplement is any food, food ingredient, vitamin, or non-food substance that is purposefully taken in addition to a normal diet in order to attain a certain performance or health benefit². Consumption of dietary supplements is a common strategy among athletes aiming to optimize recovery after training or competitions and enhance athletic performance.

Amino acids (AAs) L-arginine (Arg) and L-citrulline (Cit) are two of the most widely used supplements in recent times. Their use as nutritional supplements or dietary supplements to improve health and athletic performance has increased dramatically³, as both Arg and Cit are essential for the body’s synthesis of nitric oxide (NO) and elimination of waste products such as lactate during physical activity⁴. Because it functions as an endogenous precursor to Arg, the primary substrate of nitric oxide, Cit, a non-protein, non-essential amino acid, has attracted a lot of attention lately for its potential to improve exercise performance⁵. Commonly, citrulline is consumed as citrulline malate (CM), which is a mixture of Cit and malic acid in ratios ranging from 1:1 to 2:1^6,7.

L-Arginine

Meanwhile, Arg, a semi-essential AA, is an important precursor for NO production, synthesized through the enzyme nitric oxide synthase (NOS)⁸. Muscle tissue contains three types of NOS: neural (nNOS or NOS-1), cytokine-inducible (iNOS or NOS-2), and endothelial (eNOS or NOS-3)⁹. NO plays a critical role in various physiological functions, including vasodilation, glucose uptake, mitochondrial respiration, calcium handling, and muscle contractility, all of which contribute to enhanced exercise performance^10–12. Athletes are particularly interested in Arg supplementation for its potential to boost NO levels, providing an ergogenic advantage by supporting these functions¹³. The process involves Arg being metabolized into Cit by NOS, and liver enzymes, such as argininosuccinate lyase and argininosuccinate synthase, which recycle Cit back into Arg, creating a Cit-Arg cycle that sustains NO production¹⁴. However, due to NO’s short lifespan, its plasma concentration is typically measured indirectly via nitrite and nitrate levels¹⁴. Maintaining adequate NO levels is essential for skeletal muscle health.

Nevertheless, research on Arg supplementation has produced conflicting results concerning its potential to enhance athletic performance. Some studies suggest benefits, while others show minimal or no impact. For example, Olek et al. (2010) conducted a study examining the effects of acute Arg supplementation on anaerobic performance during repeated Wingate Anaerobic Tests (WAnTs), which are designed to measure peak power and anaerobic capacity. The investigation was conducted with a single dose of 2 g oral arginine taken 60 min before exercise assuming enhanced physical performance or altered exercise metabolism during the repeated (WAnTs)¹⁵. Their findings indicated no significant differences in peak power output following supplementation. However, the study conclusions were constrained by its limited sample size of only six participants, which likely reduced the statistical power and generalizability of the results ¹⁵. Another similar study examined the potential performance-enhancing effects of Arg supplementation using a sustained, high-intensity exercise protocol¹⁶. Their study involved a cohort of experienced, competitive cyclists tasked with completing a 20 km time trial as quickly as possible, simulating the endurance and pacing demands often encountered in real-world competitive cycling. This double-blind crossover study explored the effects of acute 3-day Arg supplementation (6 g/day) on time trial performance and oxygen consumption in six trained male cyclists. Participants consumed either Arg or placebo beverages, followed by an incremental VO₂ max test and a 20 km cycling time trial. Following the consumption of Arg, participants showed a measurable reduction in the time taken to complete the 20 km distance, indicating improved performance under conditions of sustained effort¹⁶. Results showed significantly improved time trial completion times (34 s improvement), reduced oxygen consumption, and lower blood pressure, though no difference in VO₂max was observed. In addition to the reduction in completion time, the study also observed an enhancement in the cyclists’ oxygen utilization rates, suggesting that Arg supplementation may have positively affected aerobic efficiency, potentially through the augmented availability of NO, which aids in vasodilation and blood flow to active muscles. However, despite these observed benefits in time reduction and oxygen utilization, the study found no significant changes in the participants’ maximal oxygen uptake (VO₂ max)⁴, which is a key indicator of aerobic capacity. This lack of effect on VO₂ max indicates that while Arg may improve certain physiological aspects related to endurance, it does not necessarily increase an athlete’s overall oxygen-carrying capacity or maximal aerobic power. The findings underscore the possibility that Arg may enhance performance by facilitating more efficient oxygen delivery and usage rather than by directly increasing aerobic capacity.

Citrulline malate

CM is another supplement with the potential to enhance athletic performance, offering distinct physiological benefits¹⁷. The non-essential amino acid complex has been shown to improve performance in men throughout exercise. However, these findings cannot be applied to female because of the physiological variations between the sexes^18,19. The body contains Cit, which is a strong endogenous precursor of Arg and a component of the Cit–NO cycle recycling system²⁰. Cit has been shown to aid in the clearance of ammonium from muscle tissues, a process that may help delay fatigue and sustain muscular function during prolonged or intense exercise²¹. Meanwhile, malate acts to reduce the accumulation of lactic acid, which is often associated with muscular fatigue and discomfort during high-intensity activity. By mitigating lactic acid buildup, malate supports a more favorable environment for energy production, facilitating the sustained generation of adenosine triphosphate (ATP) through aerobic pathways^21–23. Together, the components of CM may contribute to improved endurance and reduced fatigue, potentially enhancing overall performance in both aerobic and anaerobic activities²⁴. Research supporting the efficacy of CM supplementation indicates promising outcomes for enhancing performance in high-intensity, anaerobic exercise. In a study by Perez-Guisado and Jakeman²², acute CM consumption was shown to improve performance during a bench press exercise at 80% of one-repetition maximum (1RM). Participants who received CM demonstrated a greater endurance capacity, indicated by an increased time to exhaustion, suggesting that CM may help sustain muscular strength and delay fatigue under intense resistance training conditions. Similarly, in another study, Glenn et al.²⁵ 8 g CM supplementation on grip strength, vertical jump power, and Wingate anaerobic cycling conducted an investigation focusing on middle-aged female tennis players (mean age 51 ± 9 years) and found that acute CM supplementation led to a notable increase in peak power output during the Wingate Anaerobic Test, a benchmark assessment for anaerobic capacity. However, no significant differences were found for vertical jump performance or sustained anaerobic power, and the study was conducted under laboratory conditions, not real tennis match play. Collectively, these findings highlight CM’s potential to enhance power and delay fatigue, particularly during high-intensity, anaerobic activities, supporting its potential use for athletes engaged in power and strength-based sports²⁶.

The combined intake of Arg and CM could potentially enhance athletic performance more effectively than using either supplement alone. This synergistic benefit is attributed to Cit’s role in synthesizing additional Arg, which in turn elevates NO production, enhancing blood flow and possibly reducing muscle fatigue²¹. Supporting this hypothesis, Suzuki et al. (2019) conducted a study involving mixed supplementation of Arg and CM. The mixed supplements were given (1.2 g/day) over seven days on cycling performance in 24 male collegiate soccer players and found significant increases in both force exertion and fatigue resistance during a 10 min high-intensity cycling test. The improved power output during a 10 min maximal cycling test compared to placebo (242 ± 24 W vs. 231 ± 21 W, p < 0.05). Plasma concentrations of NO metabolites (NOx), CM, and Arg were also significantly elevated post-exercise in the Cit + Arg group, supporting enhanced NO production²⁷. This study provides evidence that a short period of combined supplementation can enhance both physiological and perceptual aspects of performance, particularly when administered chronically Furthermore, another study by Chen et al. (2016) explored the effects of combined Arg and CM supplementation on cognitive function among male taekwondo athletes²⁸. In a double-blind, randomized crossover design, 12 well-trained male taekwondo athletes performed three simulated matches per trial, ingesting either a combined amino acid (AA) supplement or placebo after the second match. The AA trial (0.17 g/kg BCAA, 0.05 g/kg arginine, 0.05 g/kg citrulline) maintained cognitive function, specifically premotor reaction time and secondary task performance compared to the placebo trial, where performance declined. During simulated bouts (3 rounds of 2 min), cognitive function was assessed through a validated sport-specific reaction test, revealing significant reductions in cognitive fatigue with supplementation. This improvement correlated with a reduced free tryptophan/BCAA ratio (a known marker of central fatigue), elevated NOx levels, and stable ammonia concentrations, suggesting that the combination not only supports NO-mediated blood flow and nutrient delivery but also helps buffer against neurotransmitter imbalances. While initial evidence suggests potential ergogenic benefits of combined Arg and CM supplementation, further research is needed to confirm these effects across various exercise modalities. The findings provide further support for the synergistic use of BCAA with NO precursors like Arg and CM to sustain both mental and physical performance in elite athletes during extended competition²⁸.

Accordingly, the present study aimed to investigate the acute effects of combined Arg and CM supplementation on anaerobic performance, aerobic performance, or both through high-intensity interval training (CrossFit) outcomes in healthy, trained men. Based on existing research, we hypothesized that the supplementation would enhance performance by achieving higher performance outcomes primarily in the mixed aerobic-anaerobic (CrossFit) and aerobic training protocols (Harvard Step Test) where resistance to fatigue is critical and to a lesser extent in the anaerobic protocol (Wingate Test).

Materials and methods

Study design

This study employed a randomized controlled trial design, dividing participants into three distinct exercise protocol groups: (1) CrossFit–Cindy protocol, (2) the Wingate Anaerobic Test (WAnT), and (3) the Harvard Step Test (HST). Participants allocation into the exercise groups was randomized using the randomization function in Microsoft Excel. Participants were trained individuals with a mean ± standard deviation (SD) weekly training load of 7.6 ± 3.9 h. Each participant attended three separate laboratory sessions. The initial session served exclusively for familiarization with the test protocol to minimize learning effects, without any trial performance. Subsequently in the second and the third sessions, participants attended two measurement sessions, conducted in randomized order and under double-blind conditions, where they received either the dietary supplementation or a placebo. Each participant acted as their control, performing only one exercise protocol therefore, allowing for within-subject comparisons only between supplementation or placebo conditions, but not between exercise protocols. A washout period of five to seven days was implemented between the measurement sessions to mitigate carryover effects, during which participants abstained from strenuous physical activity.

Meanwhile, randomization of the supplementation order (placebo or supplement condition) was carried out by independent laboratory personnel utilizing the randomization function in Microsoft Excel, ensuring allocation concealment. The performance outcomes were analyzed by comparing the metrics between the supplementation and placebo conditions. This trial was conducted in accordance with the principles set forth in the Declaration of Helsinki and received prior approval from the Institutional Research Ethics Committee (Approval number: TE-KEB/22/2023). The clinical trial was registered on ClinicalTrials.gov with a registration number of NCT06938126 on 22/04/2025.

Participants

An a priori sample size calculation was conducted using G*Power (version 3.1.9.7) according to previous reports²⁹, based on a large effect size, a type I error rate of 0.05, and a power of 0.8, which indicated a minimum requirement of 15 participants per exercise group for paired samples t-tests. The CrossFit exercise group consisted of 16 participants (mean ± SD: age 24.8 ± 5.0 years, height 181.0 ± 5.0 cm, body mass 78.8 ± 9.0 kg), while the Wingate Anaerobic Test (WAnT) group comprised 16 participants (mean ± SD: age 28.9 ± 7.3 years, height 177.8 ± 5.9 cm, body mass 82.9 ± 12.1 kg). Moreover, the HST group consisted of 14 participants (mean ± SD: age 22.2 ± 2.1 years, height 182.2 ± 4.9 cm, body mass 78.2 ± 13.2 kg.).

Meanwhile, eligibility criteria included being healthy, physically active men with no history of injuries or illnesses at the time of measurement or within the preceding six months. Additionally, participants who had consumed dietary supplements in the last six months were excluded to avoid confounding effects. Comprehensive written and verbal explanations regarding the nature and potential risks of the study were provided to all participants, and informed consent was obtained in written form before participation.

Dietary supplementation

The examined supplements in this study were Arg and CM. Dosages were calculated based on the manufacturer’s guidelines and the body mass of each participant. The active compound mixture consisted of a 1:1.5 ratio of CM to Arg, with a dosing routine of 0.1 g per kilogram of body mass for CM and 0.15 g per kilogram of body mass for Arg. Both supplements were provided by Nutri8 Sales Group Kft. (Biatorbágy, Hungary) in powder form.

Furthermore, to ensure blinding and consistency in taste, both the verum and placebo drinks were prepared as identical liquid mixtures comprising 100 mL of water, 150 mL of orange juice, and 150 mL of raspberry syrup. Meanwhile, the verum drink (caffeine-free power drink based on natural ingredients) only contained active supplements, with taste-masking achieved by the flavor additives. Prior to administration, liquid chromatography and mass spectrometry was conducted to quantify the exact content and confirm the proportions of each active component according to product labeling. Results showed that the active substances were present at purities above 95% and were free of any banned performance-enhancing drugs (PEDs). Participants were given a one-hour passive rest period after consuming either the supplementation or placebo to allow for absorption as applied in previous studies^4,17,30.

Anthropometric and body composition measurements

On the initial day of testing, participants underwent two anthropometric measurements. Body height was measured using an anthropometer (DKSH, Switzerland Ltd., Zurich, Switzerland), while body composition was assessed via) bioimpedance analysis (BIA) using an InBody720 device (Biospace Co., Seoul, Korea). To minimize measurement variability, participants were instructed to abstain from food and fluid intake for three hours prior and from engaging in vigorous physical activity for 24 h before the assessments.

The bioimpedance analysis provided detailed body composition data, including body fat mass, lean body mass, body fat percentage, and lean body percentage relative to total body mass^31,32. These metrics were subsequently processed for statistical analysis to evaluate the effects of supplementation on body composition parameters. The anthropometric and body composition characteristics of participants have been summarized in Table 1.

Table 1.

Anthropometric and body composition characteristics (mean ± standard deviation) of participants.

	Cindy test	Wingate test	Harvard test
	(Mean ± SD)
Number of participants	16	16	14
Age (Years)	24.8 ± 4.7	28.9 ± 7.3	22.2 ± 2.1
Body mass (Kg)	78.8 ± 10.7	82.9 ± 12.1	78.2 ± 13.2
Body height (cm)	181 ± 5.0	177.8 ± 5.9	182.2 ± 4.9
Body fat (Kg)	9.2 ± 3.3	14.4 ± 8.0	12.4 ± 8.8
Percent body fat (%)	11.6 ± 3.9	16.8 ± 7.7	14.9 ± 7.9
Skeletal muscle mass (Kg)	38.9 ± 7.9	39.2 ± 5.0	37.4 ± 4.2
Body mass index (Kg/m2)	24 ± 2.3	26. 2 ± 3.6	23.6 ± 4.0

CrossFit exercise

The CrossFit performance assessment involved the “Cindy” workout, a high-intensity protocol composed of bodyweight exercises organized in a set sequence: 5 pull-ups, 10 push-ups, and 15 squats. Participants were instructed to perform as many rounds as possible within 20 min.

Scoring was based on the total number of completed rounds and repetitions, calculated by the formula^33,34:

A full round required the successful completion of all three exercises in the specified sequence. Throughout the testing appropriate techniques were strictly monitored, such as for pull-ups, participants were required to fully extend their elbows in the suspended position and the chin had to rise to or above the bar level at the peak of the movement. For push-ups, the chest needed to touch the ground in the lowest position, with elbows fully extended at the top. Meanwhile, for squats, each repetition began from a standing position, with knees bent until the hips lowered slightly below knee level. To standardize physical readiness, a warm-up was implemented before testing. This included dynamic stretching, four exercises using resistance bands to activate key muscle groups, and one practice round of the “Cindy” workout with half the specified repetitions for each exercise. This preparatory phase ensured participants were adequately warmed up and familiar with the exercise technique, minimizing potential variability in performance due to physical readiness.

Wingate anaerobic test

The Wingate Anaerobic Test (WAnT) is employed to evaluate anaerobic performance and anaerobic capacity³⁰. This protocol involved a 30 s all-out cycling test on a bike ergometer (Monark Ergomedic 894E, Vansbro, Sweden). The test protocol began with a standardized warm-up, in which participants pedaled at 60 revolutions per minute (RPM) for five minutes without resistance. During the fourth and fifth minutes of this warm-up, two bouts of maximal pedaling were introduced, each lasting three to four seconds, to prime the participants for the intense effort required. A two-minute passive rest period followed the warm-up.

After the 30 s WAnT trial, participants were given a two-minute active recovery phase, pedaling at approximately 50 RPM with no resistance. The external resistance during the test was set to 7.5% of each participant’s body mass to ensure standardized loading across subjects.

Data on power output was recorded every second using Monark Anaerobic Test Software, an analysis tool used specifically for anaerobic tests. The key variables derived from the test included: (1) Peak Power: The highest power output achieved during the test, both in absolute terms and relative to body mass. (2) Mean Power: The average power output across the 30 s test duration, also reported in absolute and relative terms; and (3) Fatigue Index: Calculated as the percentage decline in power from the first 5 s to the last 5 s of the trial, this index reflects the rate of fatigue during the high-intensity effort. These variables were subsequently analyzed to assess the impact of supplementation on anaerobic performance and fatigue resistance.

Harvard step test

The Harvard Step Test was conducted using a 51 cm high platform, with participants stepping at a rate of 30 steps per minute (one second up, one second down) for five minutes or until exhaustion. A metronome app (Pro Metronome by EUMLab/Xiao Yixiang) on the device was used to ensure a consistent stepping pace. Participants received detailed instructions and practiced stepping on and off the box at the metronome’s rate before the test began to ensure proper timing and reduce the likelihood of pacing errors.

Upon completing the test, participants immediately sat down on a chair for the recovery phase, during which heart rate was manually measured by a designated research team member at the carotid artery. Heartbeats were counted in 30 s intervals at three specific points during the recovery period: (1) between 1 and 1.5 min, (2) 2–2.5 min, and (3) 3–3.5 min post-exercise. The test ended when participants completed five minutes of stepping or were unable to maintain the set pace for more than 15 consecutive seconds. A fitness score was then calculated based on the participant’s recovery heart rate, using the following formula from Kim et al. (2022)³⁵

whereas HR1 is the heartbeats counted after the first minute of recovery, HR2 is heartbeats counted after the second minute of recovery, and HR3 is heartbeats counted after the third minute of recovery. This score served as an index of cardiovascular fitness, with a lower recovery heart rate indicating a higher fitness level.

Complementary measurements

Blood pressure and heart rate measurements were recorded both before and two minutes after each exercise protocol. Blood pressure was measured using an Omron HEM-FL31 device (Omron HealthCare Co., Kyoto, Japan), while heart rate was monitored with a Polar Electro device (Polar Electro, Kempele, Finland).

Statistical analysis

The results are presented as mean ± standard deviation (SD), and differences are expressed as percentage changes in performance following supplementation relative to the placebo. Data were screened for outliers and tested for normality using the Shapiro–Wilk test. To assess the effects of dietary supplementation versus placebo, a paired sample t-test was conducted, with Hedges’ g used to calculate effect sizes. Considering the different outcomes of the three examined exercise modalities, crossover was performed only within supplementation conditions (and not within the exercise groups) thus, interaction effects (supplementation × exercise protocol) were not examined. Statistical significance was set at p < 0.05, and an effect size (g) greater than 0.8 was considered large. Statistical analysis was performed using SPSS version 29.0 and JASP 0.15 (JASP Team, 2021).

Results

Overall, the results revealed that the supplement condition did not statistically improve performance in the examined test protocols. The results are presented in Figs. 1, 2 and 3 and Table 3.

Fig. 1.

The diagram compares the results of the Cindy CrossFit test between two conditions supplement and placebo, based on: (A) CrossFit performance score and (B) Mean Heart Rate.

Fig. 2.

The diagram compares the results of the Wingate test between supplement and placebo, based on (A) peak power and (B) average power.

Fig. 3.

The diagram compares the results of the Harvard Step Test between supplement and placebo, focusing on Harvard Step Test Heart Rate (heart rate at different intervals) and Heart Rate mean (mean heart rate). The Harvard Step Test Heart Rate values are recorded at three time intervals: (A) 1–1:30 min, (B) 2–2:30 min, and (C) 3–3:30 min.

Table 3.

Performance-related parameters (mean ± standard deviation) for supplement and placebo conditions in all three exercises.

Variable	Unit	Supplement	Placebo	p-value (two-sided)	t-value	Hedge’s g
Cindy
Cross fit rounds		12.9 ± 3.7	12.4 ± 3.8	0.460	-0.758	0.180
Cross fit score	reps	397.6 ± 114.4	381.2 ± 113.8	0.632	-0.941	0.223
Harvard
Harvard step test Heart rate (1–1:30)	bpm	67.8 ± 8.9	68.9 ± 8.6	0.541	0.628	0.158
Harvard step test heart rate (2–2:30)	bpm	58.6 ± 8.2	59.9 ± 7.8	0.401	0.868	0.218
Harvard step test heart rate (3–3:30)	bpm	53.7 ± 7.9	54.4 ± 8.8	0.565	0.591	0.149
Time	S	277 ± 44	268 ± 59	0.216	-1.302	0.327
Harvard step test score		79.2 ± 20.0	75.4 ± 22.2	0.207	-1.327	0.334
Wingate
WAnT resistance	kg	6.2 ± 0.9	6.2 ± 0.9	0.806	-0.251	0.059
Peak power	W	1010 ± 125	986 ± 147	0.294	-1.087	0.258
Peak power	W/kg	12.5 ± 1.4	12.3 ± 1.5	0.310	-1.050	0.249
Time at peak power	ms	1687 ± 764	2813 ± 1394	0.015	2.752	0.653
Average power	W	663 ± 86	675 ± 95	0.078	1.891	0.449
Average power	W/kg	8.0 ± 0.7	8.2 ± 0.9	0.058	2.053	0.487
Power drop	%	59.5 ± 7.2	60.5 ± 6.3	0.602	0.533	0.126
Max rpm		146.5 ± 9.7	142.7 ± 11.2	0.061	-2.027	0.481

Abbreviation: CrossFit (CF), Heart Rate (HR), Beats Per Minute (bpm), Wingate Anaerobic Test (WAnT), Revolutions per Minute (rpm).

Effect of supplementation of CrossFit performance

All variables from the CrossFit measurements were normally distributed. No significant differences were found between the supplementation and placebo conditions for the primary performance measures. After the supplement condition, participants achieved a higher, but not significant performance score (397.6 points) compared to the placebo (381.2 points) (t = − 0.941; g = 0.22; p = 0.632) (Fig. 1). However, the (B) mean heart rate was nearly identical between the two conditions, with the supplement condition at 161.0 bpm and the placebo condition at 160.8 bpm (t = − 0.067; g = 0.02; p = 0.947) (Fig. 1).

Despite the absence of significant differences between supplementation and placebo, notable changes were observed between the first and second measurement days. CrossFit performance significantly improved on the second measurement day (learning effect), with performance score increasing from 364 ± 104 to 415 ± 118 (t = − 4.071; g = 0.97; p = 0.01). However, there were no significant changes in mean heart rate between the two measurement days (t = − 0.522; g = 0.12; p = 0.609). These findings suggest, as shown in Tables 2 and 3, a potential learning effect or improved performance due to familiarization with the exercise protocol, while cardiovascular responses remained consistent across the two test days. Thus, these results suggest that supplementation did not lead to meaningful improvements or changes in performance or muscle oxygenation during the CrossFit protocol.

Table 2.

Cardiovascular-related parameters (mean ± standard deviation) for supplement and placebo conditions in all three exercises.

	Cindy		Wingate		Harvard
	Supplement	Placebo	Supplement	Placebo	Supplement	Placebo
SBP pre exercise (mmHg)	133.9 ± 10.4	130.4 ± 8.4	134.1 ± 13.4	137.2 ± 13.4	134.5 ± 9.7	131.9 ± 11.4
DBP pre exercise (mmHg)	69.1 ± 9.4	70.6 ± 7.0	76.7 ± 9.9	75.4 ± 9.8	75.1 ± 7.1	72.8 ± 7.0
SBP post exercise (mmHg)	131.6 ± 10.8	133.7 ± 13.0	142.2 ± 26.1	152.6 ± 18.6	137.1 ± 15.7	140.2 ± 11.0
DBP post exercise (mmHg)	64.1 ± 14.5	63.9 ± 9.1	69.3 ± 12.0	72.4 ± 10.4	70.3 ± 8.1	73.4 ± 6.0
Resting HR (bpm)	66.3 ± 9.4	66.4 ± 9.4	73.4 ± 10.0	73.0 ± 9.6	72.3 ± 10.9	67.6 ± 8.2
HR_2min (bpm)	125.6 ± 12.0	125.2 ± 13.7	109.8 ± 12.0	110.3 ± 15.8	N/A	N/A
Mean HR (bpm)	161.0 ± 12.2	160.8 ± 15.8	146.4 ± 15.3	146.6 ± 15.7	164.1 ± 8.0	160.1 ± 7.6

Abbreviation: Systolic Blood Pressure (SBP), Diastolic blood pressure (DBP), Heart Rate (HR), Beats Per Minute (bpm), Heart rate after 2 min of recovery (HR_2min), Not applicable (N/A).

Effect of supplementation on wingate performance

The supplement condition achieved a slightly higher peak power of 1010 watts compared to 986 watts in the placebo condition (t = − 1.087; g = 0.26; p = 0.294) (Fig. 2). The average power in the supplement condition was slightly lower, but still not significant, at 663 watts compared to 675 watts in the placebo condition (t = 1.891; g = 0.85; p = 0.078) (Fig. 2). However, significant differences of moderate effect size was found in the time to peak power; shorter time was required after the supplementation compared to the placebo condition (1687 ± 764 vs 2813 ± 1394 ms, respectively) (t = 2.752; g = 0.65; p = 0.015). Maximal revolutions per minute were slightly higher (of moderate effect size) after the supplementation compared to placebo (t = -2.027; g = 0.48; p = 0.061). Results between the first and second measurement days did not differ in any variable.

Effect of supplementation on harvard performance

Fitness score in the Harvard Step Test between supplement and placebo conditions did not differ (t = − 1.327; g = 0.33; p = 0.207) (Table 3). At all intervals, the heart rate in the supplement and placebo conditions remained similar, with minor differences (Fig. 3). However, the mean heart rate under the supplement condition was slightly, but not significantly, higher (of low effect size) compared to the placebo (t = − 1.731; g = 0.44; p = 0.107). Similarly to the WAnT, neither fitness score, nor recovery rates differed between the first and second measurement days.

Discussion

Arg and CM are commonly supplemented to enhance NO production, which may improve blood flow, reduce metabolic stress, and enhance exercise performance. However, Arg alone has limited bioavailability due to rapid metabolism, while CM can more effectively raise plasma Arg levels, supporting the hypothesis that their combination may produce additive or synergistic ergogenic effects³⁶. In a study with male wrestlers, it was demonstrated that one week of combined Arg and CM supplementation reduces exercise-induced metabolic stress, improves anaerobic power, and attenuates muscle injury markers more effectively than either supplement alone³⁷. Furthermore, acute Arg supplementation is reported often fails to significantly increase NO or improve performance, whereas chronic supplementation with CM or combined Arg and CM enhances circulating NO and exercise outcomes in recreationally active and trained athletes³.

Animal studies investigating L-arginine and L-citrulline supplementation have provided foundational insights into their roles in nitric oxide (NO) production, vasodilation, and muscle metabolism. These studies demonstrate that both amino acids can enhance NO synthesis through the NOS-dependent pathway, potentially improving blood flow, oxygen delivery, and metabolic clearance in muscle tissue. However, while animal models often show favorable physiological effects, such as improved endurance, reduced fatigue, or enhanced recovery, these outcomes do not always translate consistently to human trials, highlighting the need for caution when extrapolating results³⁸. Nevertheless, a study based on randomized, double-blind, placebo-controlled crossover study examined the acute effects of CM supplementation (4.4 g) on CrossFit® performance and cardiovascular function in 21 recreationally active participants. While CM did not significantly improve workout performance (measured by rounds completed in “Cindy”), it did significantly increase time spent in heart rate zone 4 (80–90% HR max), suggesting potential cardiovascular benefits. No differences were found in post-exercise recovery time, indicating the need for further research using larger, more diverse samples and varied dosing strategies to better understand CM’s ergogenic potential⁷, highlights similar potential to the current study.

Despite these promising findings, results remain inconsistent across populations, dosages, supplementation durations, and exercise modalities. These mixed outcomes highlight the need for further investigation, particularly regarding the acute effects of combined Arg and CM on varied exercise protocols such as CrossFit, as examined in the current study. Meanwhile, the current findings did not reach statistical significance, we cautiously interpret these results as potentially reflecting limitations in study design such as the small sample size, the possibility of underdoing, and the presence of a learning effect rather than concluding a definitive absence of ergogenic effect. Moreover, Heart rate (HR) during the Harvard Step Test was measured both manually at the carotid artery and using Polar heart rate monitors. Manual measurements were required to obtain the number of beats in 30 s at the 2nd, 3rd, and 4th minutes of recovery, as per the standard fitness score formula. Although the values obtained from both methods were closely aligned and showed no systematic bias, we used the manual HR data for consistency with the original scoring method and to avoid dividing the Polar monitor values (reported in beats per minute). Another limitation of this study is that due to differing exercise modalities, between-subject comparisons were not performed, and multiple comparison corrections were not applied, which may affect the generalizability of findings across protocols.

The combined supplementation of Arg and CM did not result in significant performance improvements in the Wingate test, the Harvard Step Test, or the “Cindy” CrossFit exercise, aside from a slight advantage in reaching peak power faster during the Wingate test. The null findings included measures such as relative peak and average power, fatigue index, maximum revolutions per minute, and performance indicators in CrossFit and Harvard Test, such as rounds completed, fitness score, mean heart rate. These results may be due either to a lack of actual supplement effects or limitations in study power, as low sample sizes and modest effect sizes are common challenges in dietary supplementation trials. Meanwhile, supplementation trials feature modest participant counts, which may be insufficient for detecting small-to-moderate effect sizes.

Notably, in contrast to the present study’s null findings, Tsai et al. (2009) reported that acute L-arginine supplementation (0.1 g/kg) significantly influenced post-exercise metabolic responses in trained judo athletes, with increased glucose and insulin levels and decreased free fatty acid availability during recovery from endurance exercise. These metabolic shifts suggest that L-arginine may exert more prominent effects during the recovery phase or in endurance-based modalities rather than in high-intensity, bodyweight circuits like CrossFit. This contrast underscores the importance of considering exercise modality, supplementation timing (pre- vs. post-exercise), and metabolic context when interpreting the ergogenic potential of L-arginine ³⁹. Meanwhile, the absence of performance enhancement in this study may be related to the acute nature of L-arginine supplementation. A study by Santos et al. (2002) found that 15 days of oral L-arginine intake (3 g/day) significantly reduced muscle fatigue during isokinetic knee extensions, suggesting that chronic supplementation may be necessary to observe ergogenic effects. Additionally, the isolated resistance exercise used in their study differs from the high-intensity, full-body CrossFit protocol or lower body Wingate or Step test protocols used here, which may influence the supplement’s effectiveness^38,40.

Future research could benefit from longitudinal, multi-site trials to increase participant numbers, distributing the resource burden across labs. If the observed faster time to peak power is a true effect, it may suggest a possible interaction between the supplement’s components and the anaerobic alactacid metabolic (also known as the ATP-PC system or phosphagen system) pathway⁴¹. However, the current literature lacks mechanistic explanations linking these supplements to the alactacid system.

In dietary supplement research, underdosing often presents a significant barrier to detecting effects, especially with ergogenic aids like Arg and CM. Although the study used a dosage within the recommended range, using the highest safe dose may be more optimal even if it exceeds standard commercial doses to account for dose-dependent effects seen in preclinical models. As Cit is metabolized into Arg in the kidney, it results in a more sustained increase in Arg bioavailability than direct Arg intake. The additional step of Cit metabolism allows for a slower, prolonged increase in NO availability, which may be advantageous for muscle function, athletic performance, and recovery⁴². In this process, Cit also promotes Arg bioavailability by protecting it from enzymatic degradation before NO production occurs, creating a Cit-Arg recycling cycle⁴³. Thus, Cit’s role as an Arg precursor might provide enhanced NO utilization, potentially leading to better exercise outcomes compared to Arg supplementation alone.

One challenge in this study was determining appropriate dosing. Animal models suggest a dose–response relationship, but human studies lack robust data on ideal dosages for acute and chronic supplementation³⁴. Hence, standardized dosing based solely on body mass may not capture individual differences in muscle mass or metabolic demand, and adjusting dosage based on muscle mass may offer a more tailored approach in future studies⁴⁴.

We observed only minor effects, such as a faster time to peak power in the Wingate test, but no substantial changes in peak power, average power, fatigue index, maximal revolutions per minute, or muscle oxygen saturation. The absence of observed effects in CrossFit measures, including mean heart rate and the muscle saturation index, might be partly due to participant unfamiliarity with the exercise protocols. The observed improvement on the second day suggests a learning effect, where increased familiarity with the exercises may have improved participants’ energy utilization strategies, leading to better performance that was independent of supplementation^45,46.

Furthermore, the statistical power of the study may have been limited by the small sample size, typical of dietary supplement research. Small effect sizes in ergogenic studies are common, and this constraint may make it challenging to detect statistically significant differences, especially in short-term studies. Increasing statistical power in future studies could involve larger sample sizes, potentially achieved through multi-site, longitudinal trials to share the resource burden across labs. Additionally, studying the effects of larger dosages and incorporating middle- and long-term supplementation effects may reveal trends that acute dosing cannot capture. Past research on ergogenic aids indicates that chronic supplementation may be necessary for optimal benefits, suggesting that long-term protocols could more accurately assess the efficacy of these supplements.

Though Cit and Arg supplementation may increase blood plasma nitrite and muscle oxygenation, translating these physiological changes into improved exercise performance remains complex. For instance, Bailey et al. (2017) demonstrated that while chronic supplementation improved plasma nitrite levels and muscle oxygenation, these physiological enhancements did not necessarily translate to increased time to exhaustion⁴⁷. This highlights the need to investigate further if and how these physiological markers relate to tangible athletic outcomes.

Conclusion

In conclusion, the combined acute supplementation of 0.15 g/kg Arg and 0.1 g/kg CM appears insufficient to significantly enhance aerobic, and/or anaerobic performance in healthy, physically active men. For further investigation collaborative studies with more participants are needed to detect small-to-moderate effects and to validate the potential impact on peak power. Based on these findings and the current literature highlights the importance of including familiarization sessions in future research and suggests that Arg and CM may not provide acute performance benefits in untrained or recreational CrossFit settings. Further studies with larger, more experienced populations, varied workout protocols, and longer supplementation periods are needed to clarify L-arginine’s potential role in functional fitness performance.

Author contributions

Conceptualization—P.L., R.Z.; formal analysis, methodology, data collection—P.L., H.M., H.B., A.T., K.U., M.SU.; writing—original draft, S.S.; writing—review and editing, S.S., P.L., R.Z., A.A., R.K., K.M.H.; visualization, S.S..; supervision, P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was conducted without external funding.

Data availability

The dataset analyzed in the current study is available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This research was carried out in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Hungarian University of Sports Science (protocol code TE-KEB/22/2023; approval date: 30 June 2023).

Informed consent

Informed written consent was obtained from all participants for publication of this study.