Introduction
It has been postulated that augmenting nitric oxide (NO) synthesis through nutritional supplementation may improve muscular function, resistance to fatigue during exercise, and recovery processes after exercise (8). Thus, several supplements containing NO precursors have been marketed to recreational and competitive athletes as NO boosters claiming to improve performance (11). Nitric oxide is a labile signaling molecule that plays an important role in countless aspects of cellular and cardiovascular function. Although a comprehensive review of NO is beyond the scope of this article, enhanced NO bioavailability may favorably influence exercise performance through its effects on skeletal muscle and blood vessels (87). Nitric oxide may reduce the oxygen cost of exercise, lower the adenosine triphosphate (ATP) cost of muscle contractile force production, improve mitochondrial efficiency, and improve calcium handling (16). In addition, NO promotes vasodilation thereby reducing blood pressure and increasing blood flow, which may increase oxygen and nutrient delivery to working muscles and facilitate clearance of metabolic byproducts (36).
Until recently, L-arginine has been the chief ingredient in most NO-stimulating dietary supplements. However, oral L-arginine supplementation does not seem to effectively support NO synthesis as significant catabolism occurs by the enzyme arginase, which hydrolyzes L-arginine to L-ornithine and urea resulting in low plasma L-arginine levels (34,99). As such, L-arginine supplementation has largely yielded underwhelming effects on exercise performance (8). L-citrulline has recently garnered much attention for its potential to augment L-arginine bioavailability, NO production, and exercise performance. L-citrulline functions as an endogenous precursor to L-arginine (69) and represents an alternative and more efficient means of elevating plasma L-arginine concentrations for subsequent NO production (84). Endogenous NO synthesis occurs through the L-arginine-NO pathway, whereby L-arginine is converted to NO and L-citrulline by nitric oxide synthase (NOS) enzymes (8). Immediately after NO synthesis, NO induces vascular smooth muscle relaxation through the NO-cyclic guanosine triphosphate (cGMP) pathway leading to smooth muscle relaxation and vasodilation (34,99).
Over the past decade, L-citrulline has received considerable scientific attention examining potentially ergogenic properties for both aerobic and anaerobic exercise performance. Thus, the purpose of this article is to summarize the theoretical rationale behind L-citrulline supplementation and to comprehensively review the available scientific evidence assessing the potential ergogenic value of L-citrulline supplementation on vascular function and exercise performance in humans. In addition, research that has investigated the potential synergistic effects of L-citrulline with other dietary ingredients (e.g., arginine, antioxidants, nitrates, and branched-chain amino acids [BCAAs]) is reviewed.
L-Citrulline
It is a nonessential amino acid found primarily in watermelon, cucumbers, and other melons (34). Watermelon is the most naturally rich source of L-citrulline with amounts varying from 0.7 to 3.6 mg·g−1 of fresh weight (79). Unlike L-arginine, oral ingestion of L-citrulline is not catabolized in the gut by arginase, and enzymatic activity of argininosuccinate synthase (the enzyme that catabolizes L-citrulline) is low in enterocytes (103,104). Also, contrary to L-arginine, orally supplemented L-citrulline is not extracted from systemic circulation through hepatic clearance (81,98,102). Rather, it is transported to the kidneys where it can be directly converted to L-arginine (98,102,104). In addition, evidence suggests that L-citrulline may even suppress arginase activity, acting as a strong allosteric inhibitor, which may also play a role in upregulating L-arginine bioavailability (4,30,70).
L-citrulline is also an essential component of the urea cycle in the liver, which is responsible for ammonia elimination in the form of urea (66). During high-intensity exercise, the increased production of ammonia and inosine monophosphate in the exercising muscle has been suggested to promote muscle fatigue. High concentrations of ammonia in blood seem to increase the rate of glycolysis, resulting in the accumulation of blood lactate and increased fatigue (73). L-citrulline supplementation may assist in ammonia detoxification through the urea cycle, decrease lactate production, and enhance the aerobic utilization of pyruvate, thereby improving muscle function and attenuating fatigue (15,54,67,92).
Citrulline Malate
L-citrulline is commonly supplemented in the form of citrulline malate, and the beneficial effects may be attributed to the synergistic combination of both L-citrulline and malate at the intramuscular metabolic level (101). Malate, a tricarboxylic acid cycle intermediate, has been proposed to augment energy production and increase the rate of ATP production (6,8). In addition, malate may mitigate lactic acid production by allowing continued pyruvate production for energy utilization (100). Thus, citrulline and malate may work synergistically to increase skeletal muscle tissue perfusion and enhance the efficiency of ATP production to improve exercise performance. However, no study has compared the effects of citrulline malate to L-citrulline to delineate the potential synergistic effect of L-citrulline and malate.
Effects of L-Citrulline Supplementation on Bioactivity of Nitric Oxide
Several human studies have reported an increase in plasma citrulline and plasma arginine concentrations after oral administration of L-citrulline, citrulline malate, and watermelon juice (4,24,62,68,82,84,88,91). Furthermore, L-citrulline supplementation has shown to dose-dependently increase systemic circulation of L-arginine (68,84) and augment NO-dependent signaling more effectively when compared with an equivalent dose of L-arginine in healthy adults (84). Also, high doses of L-arginine (∼13 g) have shown to induce significant gastrointestinal complications (42), whereas L-citrulline seems to be well-tolerated at doses up to 15 g·d−1 in healthy subjects (68). Therefore, L-citrulline seems to be a more efficient intervention for increasing L-arginine bioavailability for subsequent NO production through the L-arginine-NO pathway (84).
Total nitrate and nitrite concentrations (NOx) are commonly measured as indicators of NO bioavailability in vivo, given that plasma NO is rapidly metabolized to these metabolites (34). In clinical studies, L-citrulline supplementation has shown to significantly increase NOx concentrations, along with some in vivo indices of NO bioactivity. In young and older adults with heart failure, acute ingestion of 3-g L-citrulline increased NO synthesis by ∼10-fold; however, endothelium-mediated vasodilation was not improved (53). Following 5.6-g·d−1 L-citrulline for 7 days, plasma NOx significantly increased in middle-aged men with increased arterial stiffness (74). Likewise, Morita et al. (71) reported a significant increase in NOx, along with improved brachial artery flow-mediated vasodilation, following 800-mg d−1 L-citrulline for 8 weeks in middle-aged men and women with vasospastic angina. Furthermore, L-citrulline may reduce systolic and diastolic blood pressure and seems to be more efficacious in prehypertensive and hypertensive populations (61).
In healthy populations, research investigating the efficacy of L-citrulline supplementation for augmenting NOx and vascular function is limited. A 10-g bolus of L-citrulline, in combination with 15-g whey protein, had no effect on NOx or femoral artery blood flow in healthy older men (∼72 years) (21). However, Gonzales et al. (40) showed that 6-g·d−1 L-citrulline for 14 days lowered diastolic blood pressure and increased femoral blood flow during lower-limb exercise in older men (∼71 years), whereas no changes were observed in older women. In healthy, recreationally active young men, increases in plasma arginine, citrulline, and NOx levels have been reported following 6-g·d−1 L-citrulline for 7 days (4) and ∼3.4-g·d−1 L-citrulline (in watermelon juice) for 16 days (5). Increases in plasma arginine, citrulline, and NOx levels have also been reported in trained cyclists after an acute 6-g dose of citrulline malate (88) and ∼1.47-g·d−1 L-citrulline (in watermelon juice) for 14 days (85). Yet, others have reported no change in plasma NOx during submaximal exercise after L-citrulline supplementation in recreationally active men and women (45,91). Cutrufello et al. (27) also failed to observe changes in resting flow-mediated vasodilation 60–120 minutes after consumption of either 710-ml watermelon juice or 6-g L-citrulline. However, improved muscle oxygenation has been observed after L-citrulline supplementation through near-infrared spectroscopy in recreationally active young men (4,5).
Perhaps, the strongest evidence linking oral L-citrulline supplementation to enhanced NO bioactivity is presented by Schwedhelm et al. (84). In a sample of 20 healthy subjects, oral L-citrulline administration increased plasma L-arginine concentrations, but also increased urinary cGMP and nitrate excretion, while increasing the plasma ratio of L-arginine to asymmetric dimethylarginine (ADMA); each of these effects are indicative of increased NO production and bioactivity. Furthermore, there was a strong positive correlation between the change in the L-arginine to ADMA ratio and the change in flow-mediated vasodilation (r = 0.92, p = 0.01), providing further evidence that citrulline-induced changes in plasma L-arginine translated to meaningful effects on the bioactivity of NO.
Pharmacokinetic studies of L-citrulline have shown that plasma citrulline reaches a peak concentration between 40 and 60 minutes (62,68,84). Furthermore, plasma arginine reaches a peak concentration between 80 and 120 minutes after an acute dose of L-citrulline, and plasma citrulline and arginine concentrations may remain elevated for up to 8 hours after ingestion (62,68,84). In line with these pharmacokinetic data, most studies reporting ergogenic effects of L-citrulline supplementation have provided the supplement approximately 60–90 minutes before the onset of exercise. Moinard et al. (68) reported a 10- to 100-fold increase in plasma citrulline concentration after ingestion of 2- and 15-g L-citrulline, respectively, which returned to baseline values within 5–8 hours after ingestion. These data imply that intestinal absorption of L-citrulline does not seem to be a limiting step, even at relatively high doses. Nevertheless, the pharmacokinetic parameters suggest that saturation begins to occur after the 15-g dose of L-citrulline (potentially due to saturation of its transporters or reduced renal conversion of L-citrulline to L-arginine), and the authors suggest up to a 10-g dose of L-citrulline for clinical use (68).
Despite a lack of consistent observations, numerous studies suggest that L-citrulline and/or watermelon juice supplementation can effectively increase the bioavailability of L-arginine and NOx in a wide range of populations. However, whether or not these elevations translate to improved skeletal muscle tissue perfusion remains uncertain. It has been speculated that L-citrulline supplementation may not significantly augment blood flow in young healthy volunteers because of the physiological limits of vessel compliance and intact regulatory mechanisms in otherwise healthy individuals (1). By contrast, the effects of L-citrulline on vascular function seem to be more consistently positive in clinical populations. Additional studies are needed to investigate limb blood flow and proxy measures of endothelial function in healthy, athletic populations.
Effect of Citrulline Supplementation on Time-to-Exhaustion or Time-Trial Exercise Performance
In the seminal work by Bendahan et al. (6), citrulline malate supplementation (6 g·d−1 for 15 days) seemed to significantly reduce the sensation of fatigue, increase the rate of oxidative ATP production during exercise, and increase the rate of phosphocreatine recovery after exercise. Although the exercise protocol consisted of finger flexions in a sample of men complaining of fatigue, the changes in muscle metabolism indicated that oral supplementation with citrulline malate may enhance the capacity of muscle to produce ATP aerobically and attenuate the rate of phosphocreatine breakdown. Given the potential for L-citrulline to increase NO bioavailability and improve resistance to fatigue during endurance activity, several studies have examined the effect of L-citrulline supplementation on aerobic performance in humans. Studies investigating the effect of L-citrulline supplementation on time-to-exhaustion or time-trial exercise performance are depicted in Table 1. To date, acute and loading supplementation protocols with either L-citrulline or citrulline malate have yielded rather equivocal findings.
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Table 1: Effect of citrulline supplementation during time-to-exhaustion or time-trial exercise performance.*†
Table 1-A: Effect of citrulline supplementation during time-to-exhaustion or time-trial exercise performance.*†
Contrary to the hypothesized ergogenic effects, initial work in physically active men and women by Hickner et al. (45) actually reported a reduction in treadmill time-to-exhaustion, along with higher rating of perceived exertion scores, after acute supplementation with either 3- or 9-g L-citrulline. These findings led to speculation that L-citrulline supplementation may not benefit, and may even hinder exercise performance, in young healthy adults, given that this population lacks deficiency in factors that would result in impaired blood flow. However, this study remains to be the only investigation that has reported an ergolytic effect on any exercise parameter. Subsequent L-citrulline supplementation studies have demonstrated either a neutral (5,26,27,64,85) or a positive effect (4,91) on time-to-exhaustion or time-trial exercise performance. Cutrufello et al. (27) is the only study to date that has investigated the acute timing effect of citrulline supplementation, whereby recreationally trained men and women consumed either 710-ml watermelon juice or 6-g L-citrulline either 60 or 120 minutes before a time-to-exhaustion treadmill test. However, the findings indicated no significant effect from any supplemental timing scheme on time-to-exhaustion, V̇o2peak, or anaerobic threshold. These null findings are consistent with other studies that have investigated the acute effect of a single dose of L-citrulline on time-to-exhaustion or time-trial tests (26,64). In well-trained men, Cunniffe et al. (26) administered a single 12-g dose of citrulline malate 60 minutes before a cycling protocol that consisted of a time-to-exhaustion test at 100% of the subjects' peak power. No between-trial differences were observed for performance measures, including time-to-exhaustion, peak power, mean power, average speed, cadence, distance, and work achieved. Furthermore, no between-trial differences were noted for subjective rating of perceived exertion and measures of acid-base balance (e.g., lactate, pH). Martínez-Sánchez et al (64) also demonstrated that consuming 500-ml watermelon juice enriched with L-citrulline (3.45-g L-citrulline) 120 minutes before performing a half-marathon time trial did not improve completion times or ratings of perceived exertion compared with a placebo in amateur male runners. Higher plasma lactate dehydrogenase concentrations were also elicited during the watermelon juice trial. However, watermelon juice consumption seemed to diminish the subjects' soreness perception from 24 to 72 hours after exercise, attenuate plasma lactate levels, and maintain vertical jump height after the half-marathon run.
Multiday L-citrulline supplementation protocols have also been studied with regard to time-to-exhaustion or time-trial exercise performance (4,5,85,91). After 7 days of supplementation with 6-g·d−1 L-citrulline, Bailey et al. (4) showed a significant increase in time-to-exhaustion during a moderate-intensity cycle test in recreationally active men. However, subsequent work by Bailey et al. (5) showed no effect on time-to-exhaustion or blood lactate during moderate- and severe-intensity cycle tests in recreationally active men after 16 days of enriched watermelon juice supplementation (∼3.4-g L-citrulline). Shanely et al. (85) also found no benefit on time-to-completion and mean power output during a 75-km cycle time trial after 14 days of watermelon juice (∼1.47-g L-citrulline) supplementation. However, Suzuki et al. (91) demonstrated that supplementation with 2.4-g·d−1 L-citrulline for 7 days significantly improved time-to-completion and power output during a 4-km cycle time trial. In addition, postexercise subjective feelings of muscle fatigue and concentration were significantly improved after L-citrulline supplementation compared with a placebo. Finally, additional work has shown that 28 days of supplementation with citrulline malate may attenuate exercise-induced elevations in blood lactate (54); and despite no differences observed in oxygen cost during moderate-intensity walking in young and older adults, 6-g·d−1 L-citrulline for 7 days may improve the rate of rise in V̇o2 at exercise onset in men (3).
In summary, L-citrulline supplementation has not consistently shown to improve endurance performance. A single bolus dose of L-citrulline has not shown to improve time-to-exhaustion or time-trial exercise performance. However, the current evidence suggests that approximately 7 days of continuous supplementation improves the likelihood of yielding positive outcome measures. Acute supplementation may also aid the recovery process by diminishing perceptions of muscle soreness, attenuating plasma lactate levels, and maintaining explosive power performance (i.e., vertical jump) (64). Although the current evidence is mixed, chronic dosing (>7 days) may improve measures of endurance performance (i.e., time-to-exhaustion or time-to-completion).
Effect of Citrulline Supplementation on High-Intensity Exercise Performance
Few studies have examined the acute effect of L-citrulline supplementation on high-intensity exercise performance such as cycling sprints and vertical jump power (depicted in Table 2) (26,38,93). Glenn et al. (38) examined vertical jump and Wingate anaerobic cycling performance 60 minutes after the ingestion of 8-g citrulline malate in masters-aged (51 years) female tennis players. Compared with a placebo, no differences were found between trials for peak or mean vertical jumping power. However, for the 30-second Wingate, peak power and explosive power were significantly greater after consuming citrulline malate compared with the placebo. Yet, anaerobic capacity and the ability to sustain anaerobic power did not differ between trials.
Table 2: Effect of citrulline supplementation during high-intensity exercise performance.*†
Other investigations have examined the effect of acute L-citrulline supplementation on repeated cycling sprint performance. Tarazona-Díaz et al. (93) administered either natural watermelon juice containing 1.17 g of L-citrulline, enriched watermelon juice containing a total of 6 g of L-citrulline, or a placebo 60 minutes before a cycling test consisting of eight 30-second maximum effort intervals, separated by 60 seconds of rest. In this sample of recreationally active men, no significant differences were observed for revolutions per minute, ratings of perceived exertion, or blood lactate. However, both watermelon juice trials seemed to attenuate feelings of muscle soreness 24 hours after the exercise bout. No differences between trials were noted for muscle soreness at 48 hours post. Cunniffe et al. (26) investigated the effect of 12-g citrulline malate supplementation on maximal cycling sprints in well-trained men. Citrulline malate or placebo was provided 60 minutes before performing ten 15-second maximal cycle sprints with 30-second rest intervals. No differences between trials were noted for peak power, mean power, fatigue index, ratings of perceived exertion, blood lactate, or acid-base balance. Based on the intermittent sprint protocols used in these studies, supplementation with watermelon juice, L-citrulline, or citrulline malate does not seem to provide acute ergogenic benefits.
Limited evidence exists regarding the effect of multiday L-citrulline supplementation on high-intensity exercise performance and training-induced adaptations. As part of the study by Bailey et al. (4), recreationally active men performed a 60-second all-out sprint after supplementing with 6-g·d−1 L-citrulline for 6 days earlier. Peak and mean power output, along with total work completed, was significantly greater during the 60-second all-out sprint after L-citrulline supplementation compared with a placebo. Buckinx et al. (14) supplemented the diets of inactive, older (>60 years) dynapenic-obese men and women with 10-g·d−1 L-citrulline in conjunction with a 12-week, 3-d·wk−1 high-intensity interval training (HIIT) program on an elliptical. Compared with a placebo group, those supplementing with L-citrulline demonstrated greater improvements in the “Timed Up and Go” test and upper-limb muscle strength (measured using hand dynamometer) after the training intervention. However, no significant differences were noted between groups for other assessments including body composition, lower-limb muscle strength and power, muscle quality, 6-minute walking test, balance, chair stand test, and step test. Supplementation with L-citrulline for multiple days (≥6 days) may enhance performance during an all-out cycling sprint in recreationally active men, and when combined with HIIT training, supplementation may provide benefit to some indices of muscle function in otherwise healthy, dynapenic-obese older adults. Discernibly, more researched is warranted to explore the potential benefits of L-citrulline supplementation on high-intensity performance and training adaptations.
Effect of Citrulline Supplementation on Strength Exercise Performance
As previously mentioned, Bendahan et al. (6) investigated the effects of citrulline malate on muscle energetics during repetitive exercise based on previously reported antiasthenic effects of citrulline malate. Although the results indicated that citrulline malate reduced subjective fatigue while increasing oxidative ATP production and phosphocreatine recovery, the exercise task tested (repeated finger flexions) has limited applicability to traditional resistance exercise. Several years later, Perez-Guisado and Jakeman (78) conducted a crossover trial in which the effects of citrulline malate were evaluated within the context of a resistance exercise test protocol with greater ecological validity for athletic populations. Subjects ingested 8-g citrulline malate or a placebo treatment 60 minutes before resistance exercise. The testing protocol consisted of 4 exercises targeting the pectoralis major muscle group, for a total of 16 sets taken to volitional failure. Results indicated that citrulline malate significantly increased repetitions completed before failure, particularly in later sets, and significantly reduced subjective muscle soreness 24 and 48 hours after exercise.
Multiple subsequent studies have seemed to support the findings of Perez-Guisado and Jakeman (78), indicating that citrulline malate enhances muscular endurance during fatiguing resistance exercise. For example, a study in resistance-trained men evaluated the effects of citrulline malate (8 g, ingested 60 minutes before exercise) on repetitions to fatigue during lower-body exercise (101). The testing protocol consisted of 3 sets each of leg press, hack squat, and leg extension, performed with 60% of the 1 repetition maximum (1RM) load. Although citrulline malate did not significantly influence blood lactate, heart rate, or blood pressure, results indicated that citrulline malate significantly increased repetitions to fatigue. The same research group published a follow-up study (100), in which 8-g citrulline malate was also shown to significantly improve upper-body repetitions to fatigue (chin-ups, reverse chin-ups, and push-ups) without significantly affecting blood lactate levels. Although the previously mentioned studies were conducted using male samples, results have also been replicated in female samples. Glenn et al. (39) tested the effects of 8-g citrulline malate on both upper-body (bench press) and lower-body (leg press) exercise in resistance-trained women using 80% 1RM loads. Citrulline malate did not affect heart rate responses to exercise but did significantly increase upper- and lower-body repetitions to fatigue while reducing perceived exertion during upper-body exercise. The same research group also investigated the effect of citrulline malate in female masters tennis players (38). Compared with a placebo, 8-g citrulline malate enhanced maximal grip strength, average grip strength, and both peak power and explosive power during a Wingate test. By contrast, peak vertical power, average vertical power, and the ability to sustain power during the Wingate test were not significantly affected.
Although numerous studies have reported statistically significant improvements in resistance exercise performance after citrulline supplementation, many others have not. For example, Gonzalez et al. (41) assessed the effects of 8-g citrulline malate on resistance exercise performance in resistance-trained men. The performance assessment included 5 sets of 15 repetitions on the bench press exercise, using a 75% 1RM load with 2 minutes of rest between sets. Citrulline malate did not significantly enhance repetitions to fatigue, peak or mean bench press power, focus, energy, fatigue, or muscle swelling. Farney et al. (31) studied the effects of 8-g citrulline malate on leg extension performance after completion of a high-intensity exercise circuit consisting of squats, lunge jumps, squat jumps, and lateral jumps. Citrulline malate did not significantly affect total work, peak torque, peak power, rate of fatigue, or blood lactate accumulation. Finally, Chappell et al. conducted 2 studies, each investigating German Volume Training protocols for either upper-body (18) or lower-body (17) exercise. Both protocols encouraged subjects to attempt to complete 10 sets of 10 repetitions; loads included 80% of 1RM for the barbell curl exercise (18) and 70% of peak concentric force for leg extensions (17). Although 8-g citrulline malate successfully reduced soreness for the bicep curl exercise (18), it surprisingly increased soreness after leg extension (17). In both studies, citrulline malate was ineffective for enhancing indices of resistance exercise performance (17,18).
In light of mixed findings from individual studies assessing the ergogenic potential of L-citrulline for strength performance (depicted in Table 3), a recent meta-analysis sought to perform a pooled analysis of the literature evaluating the effects of acute L-citrulline supplementation on high-intensity strength and power outcomes (96). After pooling results from 13 samples including 198 total subjects, the overall effect of supplementation on performance was small but statistically significant, with a pooled standardized mean difference (Hedges' G) of 0.20. L-citrulline doses studied typically ranged from 3 to 6 g, while the most common dosing strategy for traditional resistance exercise protocols was 8 g of citrulline malate, ingested 60 minutes before exercise. Although the small number of overall studies was a limitation with regard to testing for potential moderating effects, moderator analyses did not suggest that study results were significantly impacted by sex, training status, supplement form, musculature tested, type of exercise outcome, modality of exercise, or funding source. However, the authors of the meta-analysis indicated that the results should be interpreted as preliminary in nature, as the body of L-citrulline literature pertaining to strength and power outcomes was small and rapidly growing at the time of its publication. As such, our understanding of L-citrulline's ergogenic potential for strength exercise performance is almost certain to improve in coming years. Assessment of the current literature would tentatively suggest that citrulline may yield small but statistically significant improvements for strength exercise performance, but future studies are needed to reinforce this conclusion and to more closely investigate the potential moderating effects of sex, supplement form, citrulline dose, and other key study characteristics.
Table 3: Effect of citrulline supplementation during strength exercise performance.*†
Table 3-A: Effect of citrulline supplementation during strength exercise performance.*†
Secondary to direct, acute performance effects, L-citrulline supplementation may facilitate long-term adaptations to resistance training through anabolic effects. Nitric oxide, regardless of its source, is believed to play a role in skeletal muscle hypertrophy by mediating satellite cell activation (2). This link between NO and muscle hypertrophy is supported by multiple rodent studies. In 1 study (86), drinking water was supplemented with N(G)-nitro-L-arginine methyl ester (L-NAME) to block endogenous NO production in Sprague-Dawley rats undergoing skeletal muscle overloading. Compared with the control condition, the addition of L-NAME substantially inhibited several of the muscular adaptations to overloading, including hypertrophy. In a separate rodent study (57), an NO donor (isosorbide dinitrate) was orally administered to aging mice that were participating in voluntary exercise. By the end of the intervention, quadriceps muscle fiber diameter was greater in mice in the exercise + isosorbide dinitrate condition compared with all other groups (exercise only, isosorbide dinitrate, and control).
Although there is some evidence to suggest that NO per se has potential to promote hypertrophic responses to skeletal muscle loading, Breuillard et al. (13) suggest that citrulline may uniquely promote muscle protein accretion, presumably due to its limited uptake by the liver and its effects on the urea cycle. As reviewed by Papadia et al. (75), both animal and human studies have indicated that L-citrulline positively impacts protein synthesis. For example, a crossover trial conducted with 8 human subjects found that L-citrulline ingestion increased the fractional synthesis rate of muscle protein to a greater extent than a nonessential amino acid mixture after 3 days of low protein intake (51). Similarly, oral L-citrulline supplementation increased nitrogen balance by 57% compared with a placebo treatment in 10 healthy human subjects (82). A recent study by Bouillanne et al. (12) found no effect of L-citrulline supplementation on whole-body or liver protein synthesis in malnourished older adults, but supplementation increased lean mass and appendicular skeletal muscle mass in female subjects nonetheless.
Although the mechanisms underlying the purported anabolic effects of citrulline are not fully elucidated, it is believed that citrulline-induced increases in protein synthesis are possibly mediated by the mammalian target of rapamycin pathway (13). In addition, as reviewed by Papadia et al. (75), calcium-induced activation of the Akt pathway seems to be dependent on NOS activity (29), and evidence suggests that nitrite increases proliferation of myocyte precursor cells through a mechanism that is independent of NO and cGMP (95). However, it is important to note that conflicting results have been reported. For instance, Churchward-Venne et al. (21) found that adding 10 g of L-citrulline to 15 g of whey protein did not increase muscle protein synthesis more than adding a 10-g mixture of nonessential amino acids to the same dose of whey.
Finally, there are 2 longitudinal human studies assessing the hypertrophy-promoting potential of L-citrulline supplementation. In a study by Figueroa et al. (33), L-citrulline (6 g) was compared with a placebo treatment in postmenopausal women completing a whole-body vibration training program targeting the leg musculature. After the 8-week intervention, the addition of L-citrulline led to more favorable changes in leg lean mass index, and slightly (but not statistically significant) better changes in leg muscle strength, when compared with vibration training alone. Hwang et al. (47) conducted a longitudinal resistance training study in which muscular adaptations to an 8-week resistance training program were assessed in healthy, resistance-trained men. Throughout the intervention, subjects supplemented with L-citrulline plus glutathione (CIT + GSH; 2-g·d−1 CIT + 200-mg·d−1 GSH), citrulline malate (2 g·d−1), or a placebo. At the 4-week point, lean mass changes were slightly greater in CIT + GSH compared with placebo, but measurements taken at the conclusion of the 8-week intervention revealed no significant benefits of CIT + GSH or citrulline malate for performance or body composition. However, it should be noted that the L-citrulline doses used in this study were fairly low, and the resistance training program did not seem to induce meaningful body composition adaptations. As such, it is difficult to rule out the possibility that larger doses of citrulline, in conjunction with a training and nutrition program more conducive to hypertrophy, might confer some benefit.
At this time, the body of literature pertaining to L-citrulline and strength exercise is small but rapidly growing. Evidence currently supports the tentative conclusion that citrulline doses over 3 g confer small but statistically significant improvements for high-intensity strength and power performance. Although there is a mechanistic basis and preliminary evidence to suggest that citrulline may promote muscle protein accretion, the singular longitudinal study providing L-citrulline supplementation in conjunction with resistance training in healthy, resistance-trained subjects failed to observe meaningful benefits for performance or body composition. Future research should continue to elucidate the effects of citrulline supplements on strength and body composition, with a particular focus on longitudinal interventions spanning several weeks.
Finally, it is prudent to note that nitrate, through the NOS-independent pathway of NO production, might also be a suitable NO precursor for the purpose of enhancing high-intensity strength and power performance. As reviewed by Lundberg et al. (58), the NOS-independent pathway recycles nitrate and nitrite to form NO, and this pathway is stimulated by hypoxia and acidosis, which characterize the local metabolic environment during high-intensity exercise. Although the endogenous NOS-dependent pathway is a contributing source of nitrate and nitrite, dietary nitrate can also be consumed through food and supplements. Emerging studies are beginning to suggest that dietary nitrate, often consumed in the form of beetroot juice, may indeed improve indices of strength and power and delay fatigue during resistance-type exercise (22,23,72,94). However, there is still a limited number of studies evaluating the effects of beetroot juice and other nitrate sources on high-intensity strength and power protocols with high ecological validity. More research is needed to conclusively determine whether dietary nitrate sources enhance strength and power in performance tests that approximately reflect real-world training protocols or the demands of athletic competition to compare the efficacy of nitrate and L-citrulline in such tasks and to evaluate the possibility of complementary effects when both ingredients are coingested.
Coingestion of L-Citrulline and Other Dietary Supplements
Multi-ingredient preworkout supplements containing a blend of ingredients, such as caffeine, creatine, beta-alanine, amino acids, and NO precursors, have increased in popularity and are regularly marketed to the public as a way to enhance exercise performance (43). L-citrulline is among the most common ingredients found in commercially available multi-ingredient preworkout supplements (49). A recent review of the top commercially available preworkout supplements indicated that L-citrulline was found in 71% of products with an average listed amount of 4.0 ± 2.5 g (49). Although some studies have shown that multi-ingredient preworkout supplements containing citrulline can enhance exercise performance (7,83), it is difficult to delineate the ergogenic value of citrulline under these circumstances. Currently, there are limited data regarding the effects of citrulline in combination with other potentially performance enhancing ingredients. The objective of this section is to discuss the research that has investigated the potential synergistic effects of citrulline with other dietary ingredients.
Citrulline + Arginine
The combined effect of L-citrulline and L-arginine supplementation may have a more beneficial effect than a single dose of either amino acid alone. In a rodent model, acute coingestion of L-citrulline and L-arginine has shown to result in a greater and more rapid increase in plasma arginine, NOx, and peripheral artery blood flow as compared to either amino acid in isolation (70). In healthy men, a combination of 1-g·d−1 L-arginine and 1-g·d−1 L-citrulline also seems to increase plasma arginine levels more effectively than either 2-g·d−1 L-arginine or 2-g·d−1 L-citrulline (90). One possible explanation for this synergistic effect might be due to the inhibiting effect of L-citrulline on arginase (4,30,70). A recent study in male soccer players also showed that supplementing with L-citrulline and L-arginine (1.2 g·d−1 of each for 7 days) significantly improved power output and subjective perceptions of “leg muscle soreness” and “ease of pedaling” during a 10-minute high-intensity cycling test, along with elevated concentrations of plasma citrulline, arginine, and NOx (89). Further investigations into the potentially synergistic effect of L-citrulline and L-arginine are warranted. Future studies should investigate the effect of the combination of L-citrulline and L-arginine vs. each amino acid in isolation.
Citrulline + Antioxidants
It has long been known that antioxidants modulate the production and bioactivity of NO. For instance, antioxidants present in pomegranate juice have been shown to prevent the degradation of NO by reactive species (48), and polyphenols facilitate the reduction of nitrite to NO in the gut (77). Similarly, ascorbic acid has been shown to protect tetrahydrobiopterin, a critical cofactor involved in the conversion of arginine to NO through NOS, from oxidation. As a result, this protective effect on tetrahydrobiopterin thereby facilitates NO synthesis (44). This relationship between antioxidants and NO may explain, in part, favorable effects of pomegranate-based supplements on indices of blood flow (80,97) and comparatively more pronounced effects from beetroot juice supplementation compared with nitrate-matched amounts of sodium nitrate (35).
Watermelon juice is a source of both citrulline and antioxidants, but the naturally occurring L-citrulline content in a 500- to 700-ml serving of watermelon juice is typically under 2 g. As such, several studies have investigated the ergogenic potential of citrulline-fortified watermelon juice, raising the total citrulline content to roughly 3–6 g of total citrulline. As previously noted, Tarazona-Díaz et al. (93) investigated the effects of both regular watermelon juice (1.17 g of citrulline) and citrulline-enriched watermelon juice (with an additional 4.83 g of citrulline, for a total of 6 g) on cycling revolutions per minute, perceived exertion, subjective soreness, blood lactate, and heart rate recovery in response to an intermittent sprint test on a cycle ergometer. Results indicated that both watermelon juice treatments significantly reduced subjective soreness 24 hours after exercise, while no other statistically significant effects were observed.
The effects of citrulline-enriched watermelon juice on muscle soreness were also studied by Martinez-Sanchez et al. (63), in addition to blood markers of muscle damage and maintenance of force production after resistance exercise. The exercise protocol used the half-squat exercise, with subjects completing 8 sets of 8 repetitions each. Results of the study showed that beverages containing L-citrulline and antioxidants generally had favorable effects on the attenuation of muscle damage, soreness, and force reductions after fatiguing exercise. Notably, results were more favorable for citrulline-enhanced watermelon juice (3.3-g citrulline) compared with regular watermelon juice (0.5-g citrulline), and results were even more favorable when antioxidant-rich pomegranate juice (containing 22.0-mg ellagitannins) was added to the citrulline-enriched watermelon juice. As was previously discussed, the same laboratory group conducted a separate study (64) in which citrulline-enriched watermelon juice (3.45-g citrulline) or a placebo treatment was consumed before a half-marathon race, with outcome measures including jump height, heart rate, perceived exertion, subjective soreness, and a variety of blood biomarkers. Although watermelon juice did not enhance time-trial performance, it significantly reduced subjective muscle soreness from 24 to 72 hours after the half-marathon. Immediately after the race, the watermelon juice also yielded lower blood values of lactate and glucose and higher values of lactate dehydrogenase and L-arginine in comparison with placebo. Finally, jump height was reduced after race completion in the placebo condition, but this reduction in jump height was not observed in the watermelon juice condition.
Despite these favorable findings, other studies have failed to show that the synergistic relationship between NO and antioxidants translates to physiologically meaningful benefits. As noted previously, 3 studies assessing the effect of citrulline-based watermelon supplements all failed to observe significant ergogenic effects. Shanely et al. (85) found that watermelon puree (1.47-g·d−1 L-citrulline for 2 weeks) increased postexercise citrulline, arginine, and nitrate levels, while reducing perceived exertion during the 75-kg cycling time trial. However, the watermelon puree treatment did not enhance time-trial performance or influence systemic biomarkers of inflammation or innate immune function. Although this study was limited by a low daily dose of citrulline, other studies with higher dosages have also failed to detect meaningful benefits of L-citrulline supplementation. Bailey et al. (5) implemented a crossover trial in which subjects consumed watermelon juice (3.4-g citrulline) or a placebo treatment for 16 days, followed by exercise testing. Although watermelon juice increased plasma nitrite concentrations and improved muscle oxygenation during moderate-intensity cycling exercise, it also increased resting systolic blood pressure and failed to improve time to exhaustion during high-intensity exercise. Finally, Cutrufello et al. (27) evaluated the effects of L-citrulline (6 g) or watermelon juice (1-g L-citrulline) on blood flow and a variety of exercise outcomes. Compared with a placebo, neither treatment significantly improved chest press repetitions, time to exhaustion, V̇o2 max, anaerobic threshold, or flow-mediated vasodilation.
As an important antioxidant and free radical scavenger, it has been speculated that glutathione in its chemically reduced form (GSH) has the capacity to improve the bioactivity of NO. In addition, GSH seems to play a role in NO synthesis, and NO can react with GSH to form S-nitrosoglutathione, which is believed to act as a temporary reservoir that stabilizes NO, protects it from oxidative damage, and allows for a more sustained release of NO (47,76). Data evaluating acute administration indicate that a synergistic relationship between GSH and NO exists, such that GSH improves markers of NO synthesis after L-citrulline supplementation (65). Nitrate concentration measured in cells incubated with CIT + GSH was higher than control cells, whereas nitrate levels were not significantly altered in cells treated with L-citrulline or GSH alone. Furthermore, rodents were infused with L-citrulline, CIT + GSH, or a control infusion, and plasma NOx levels were assessed for the next 4 hours. Compared with L-citrulline or the control condition, CIT + GSH infusion caused a more robust increase in plasma NOx levels. Finally, the results were translated to humans undergoing resistance exercise, after 7 days of supplementation with a cellulose placebo, L-citrulline (2 g·d−1), GSH (1 g·d−1), or CIT + GSH (2-g·d−1 CIT + 200-mg·d−1 GSH). Only the CIT + GSH treatment resulted in significantly greater postexercise plasma nitrite and NOx elevations compared with the placebo treatment (65).
As previously discussed, a follow-up study assessed the effects of prolonged supplementation on adaptations to resistance training (47). Seventy-five resistance-trained men were randomized to groups ingesting CIT + GSH (2-g·d−1 L-citrulline + 200-mg·d−1 GSH), citrulline malate (2 g·d−1), or a placebo. Treatments were ingested 60 minutes before exercise on training days and with breakfast on nontraining days. Training involved 8 weeks of supervised, periodized resistance training consisting of 2 upper-body and 2 lower-body workouts per week. After 4 weeks of training, only CIT + GSH increased lean mass more than placebo. However, at the conclusion of the 8-week training program, strength and body composition outcomes did not differ among groups. At this time, findings related to the combination of L-citrulline and GSH are preliminary in nature. Results suggest that GSH synergistically facilitates NO production when combined with L-citrulline; however, this may not influence chronic adaptations to resistance training. Notably, interventions to date have used fairly low doses of L-citrulline (2 g·d−1) when used in combination with GSH; future research should evaluate other dosing combinations of L-citrulline and GSH.
In summary, there is a strong mechanistic basis underlying the synergistic relationship between antioxidants and L-citrulline. Although there are instances in which the combination of antioxidants and L-citrulline has failed to yield significant ergogenic effects, adding an antioxidant source to L-citrulline seems to have either a neutral or positive effect on its ergogenic capacity. The presence of both positive and null performance effects of L-citrulline plus antioxidants in the literature likely relates to the wide variation of key study characteristics present within the literature, such as dosing strategies, performance tests, and sample characteristics.
Citrulline + Nitrates
Dietary nitrate (NO3−) is another ergogenic nutritional supplement that is widely used to improve cardiovascular health and sports performance (16,50,52). Dietary nitrates serve to enhance NO bioavailability and vascular function through a distinct NOS-independent pathway. Nitric oxide synthesis can occur through the reduction of nitrate to NO through the nitrate-nitrite-NO pathway (25,58). Thus, it can be suggested that supplementation with a combination of NO precursors (i.e., L-citrulline and nitrate) may improve endothelial function, skeletal muscle tissue perfusion, and exercise performance in humans; however, this hypothesis has yet to be fully elucidated. Le Roux-Mallouf et al. (56) examined the acute effect of nitrate and L-citrulline supplementation (1,200 mg [19.4 mmol] of nitrate and 6 g of L-citrulline, respectively) on postischemic microcirculation hyperemia in healthy men and women at rest. Near-infrared spectroscopy assessment indicated significantly larger increases in total hemoglobin and oxyhemoglobin concentrations for the thigh and calf muscles after nitrate/L-citrulline supplementation compared with a placebo. The larger postocclusive changes in total hemoglobin and oxyhemoglobin suggest a greater postischemic vasodilation, which may be due, in part, to increased NO availability after supplementation. In a follow-up study by Le Roux-Mallouf et al. (55), healthy men performed a thigh ischemia-reperfusion test followed by submaximal isometric knee extensions after consumption of either a nitrate-rich beetroot juice (520-mg nitrate) alone or in combination with either L-citrulline (6 g) or L-arginine (6 g). Compared with a placebo trial, the nitrate alone and nitrate/L-citrulline intake exerted positive effects on postischemic vasodilation assessed by near-infrared spectroscopy. However, the nitrate/L-citrulline intake was not superior to nitrate alone indicating a lack of a synergistic effect of the NO precursors compared with nitrate in isolation. Furthermore, no significant effect was observed for muscle and cerebral oxygenation, peripheral and central mechanisms of neuromuscular fatigue, or total number of knee extensions during exercise after any supplement trial. The authors speculate that NO bioavailability in healthy subjects may not be the limiting factor for tissue perfusion and oxygenation during submaximal knee extension repetitions to fatigue. Nevertheless, future research is required to delineate the role of each dietary ingredient by comparing the effects to nitrates and L-citrulline in isolation and in combination. In addition, studies should continue to evaluate the effect of supplementing with a combination of NO precursors on the ability to enhance blood flow, skeletal muscle tissue perfusion, and physical performance.
Citrulline + Branched-Chain Amino Acids
Ingestion of BCAAs before exercise has been purported to exert positive effects on muscle soreness and exercise-induced muscle damage (37). In addition, BCAAs serve as a competitive inhibitor of tryptophan, the precursor for serotonin synthesis, and may play a role in reducing central fatigue during prolonged exercise by reducing cerebral serotonin synthesis (9,10,32). Notably, Sureda et al. (88) suggested that citrulline malate supplementation may increase the use of branched chain amino acids as fuel during exercise. Recently, a series of studies have investigated the effect of ingesting a supplement containing a combination of BCAA, L-arginine, and L-citrulline (0.085- to 0.17-g·kg−1 BCAA, 0.05-g·kg−1 arginine, and 0.05-g·kg−1 citrulline) 60 minutes before exercise (19,20,46,105). The combination supplement may alleviate exercise-induced central fatigue in athletes and help maintain cognitive function (i.e., execution of motor skills and reaction time) after competitive athletic endeavors (19,105). Furthermore, Cheng et al. (20) showed that ingestion of the combination supplement improved time-trial performance during 2 consecutive days of testing (5,000- and 10,000-m run, respectively) in male and female endurance trained runners. Hsueh et al. (46) also showed that the combination supplement improved average 50-m swim time in young male and female swimmers during an 8 × 50-m swim protocol. These studies (19,20,46,105) also report a significantly lower tryptophan/BCAA ratio after supplementation, with no differences observed between trials for plasma concentrations of ammonia. Previous studies have shown that BCAA supplementation, in isolation, typically fails to show ergogenic benefits on a single bout of exercise and results in an increase in plasma concentrations of ammonia (59,60). Given the effects of L-citrulline on urea cycle function, the addition of citrulline/arginine in these studies may have alleviated the excess ammonia accumulation and allowed for improved performance (20). Future research should investigate this hypothesis by examining the effect of BCAA, in isolation, compared with the combination supplement.
Future Directions
Overall, short-term supplementation of L-citrulline has shown to be safe and well-tolerated. However, up to 15% of subjects have reported stomach discomfort as a side effect after ingestion (26,78). On the other hand, Moinard et al. (68) noted that L-citrulline supplementation did not induce gastrointestinal distress at doses up to 15 g. A major limitation of the current evidence is the short-term nature of most intervention studies; thus, the safety and efficacy of long-term L-citrulline supplementation requires further investigation.
Given the positive effects observed from some investigations, further trials should be performed using both acute and loading doses of L-citrulline or citrulline malate on exercise performance in a variety of populations who may benefit from supplementation (i.e., recreational athletes, resistance-trained populations, aerobic and anaerobic athletes, bodybuilders, older adults, clinical populations, etc). It also remains unclear whether the observed effects in studies administering citrulline malate are mediated by malate, L-citrulline, or both. Currently, there is no study comparing the effects of L-citrulline vs. citrulline malate to delineate the potential synergistic effect of each component. Future studies should also continue to investigate the vascular effects of L-citrulline supplements and its potentially synergistic effects with other ingredients.Practical Applications
L-citrulline, a nonessential amino acid found primarily in watermelon, has recently garnered much attention for its potential to augment L-arginine bioavailability and NO production and thereby enhance exercise performance. Over the past decade, numerous studies have investigated the ergogenic properties of L-citrulline for both aerobic and anaerobic exercise performance. Collectively, oral L-citrulline supplementation has shown to increase plasma citrulline, arginine, and NOx. Although blood flow enhancement is a proposed mechanism for the ergogenic potential of L-citrulline, evidence supporting acute improvements in vasodilation and muscle tissue perfusion after supplementation is scarce and inconsistent. Nevertheless, several studies have reported that L-citrulline supplementation can enhance exercise performance and recovery. Based on the current evidence, chronic dosing (>7 days) seems to be more effective than an acute single-dose protocol for enhancing exercise performance. The minimum effective dose seems to be ∼3 g·d−1 of L-citrulline, while the maximum effective dose may be as high as 10–15 g·d−1. Citrulline malate products often provide a 1:1 or 2:1 ratio of citrulline to malate, although the literature to date has often failed to specify the labeled ratio of the product used. Studies reporting ergogenic effects with citrulline malate tend to provide a 6- to 8-g dose, which would provide at least 3 g of L-citrulline at either ratio. It is unclear how malate would provide a robust contribution to the ergogenic effect of citrulline malate; so, a 2:1 citrulline-to-malate ratio may be favored. Finally, L-citrulline ingestion 60–90 minutes before the onset of exercise seems to most reliably enhance performance, whether ingested as L-citrulline or citrulline malate. Future studies should continue to investigate the effects of both acute and chronic supplementation with L-citrulline and citrulline malate on markers of blood flow and exercise performance and should seek to elucidate the mechanism underlying such effects.
Acknowledgments
A.M. Gonzalez and E.T. Trexler declare no conflicts of interest.
References
1. Allerton T, Proctor D, Stephens J, et al. L-citrulline supplementation: Impact on cardiometabolic health. Nutrients 10: 921, 2018.
2. Anderson JE. A role for nitric oxide in muscle repair: Nitric oxide–mediated activation of muscle satellite cells. Mol Biol Cell 11: 1859–1874, 2000.
3. Ashley J, Kim Y, Gonzales JU. Impact of L-citrulline supplementation on oxygen uptake kinetics during walking. Appl Physiol Nutr Metab 43: 631–637, 2018.
4. Bailey SJ, Blackwell JR, Lord T, et al. L-citrulline supplementation improves O2 uptake kinetics and high-intensity exercise performance in humans. J Appl Physiol 119: 385–395, 2015.
5. Bailey SJ, Blackwell JR, Williams E, et al. Two weeks of watermelon juice supplementation improves nitric oxide bioavailability but not endurance exercise performance in humans. Nitric Oxide 59: 10–20, 2016.
6. Bendahan D, Mattei J, Ghattas B, et al. Citrulline/malate promotes aerobic energy production in human exercising muscle. Br J Sports Med 36: 282–289, 2002.
7. Bergstrom HC, Byrd MT, Wallace BJ, Clasey JL. Examination of a multi-ingredient preworkout supplement on total volume of resistance exercise and subsequent strength and power performance. J Strength Cond Res 32: 1479–1490, 2018.
8. Bescos R, Sureda A, Tur JA, Pons A. The effect of nitric-oxide-related supplements on human performance. Sports Medicine 42: 99, 2012.
9. Blomstrand E, Hassmén P, Ek S, Ekblom B, Newsholme E. Influence of ingesting a solution of branched-chain amino acids on perceived exertion during exercise. Acta Physiol Scand 159: 41–49, 1997.
10. Blomstrand E, Hassmen P, Ekblom B, Newsholme E. Administration of branched-chain amino acids during sustained exercise-effects on performance and on plasma concentration of some amino acids. Eur J Appl Physiol Occup Physiol 63: 83–88, 1991.
11. Bloomer RJ. Nitric oxide supplements for sports. Strength Cond J 32: 14–20, 2010.
12. Bouillanne O, Melchior JC, Faure C, et al. Impact of 3-week citrulline supplementation on postprandial protein metabolism in malnourished older patients: The Ciproage randomized controlled trial. Clin Nutr 38: 564–574, 2019.
13. Breuillard C, Cynober L, Moinard C. Citrulline and nitrogen homeostasis: An overview. Amino Acids 47: 685–691, 2015.
14. Buckinx F, Gouspillou G, Carvalho L, et al. Effect of high-intensity interval training combined with L-citrulline supplementation on functional capacities and muscle function in dynapenic-obese older adults. J Clin Med 7: 561, 2018.
15. Callis A, de Bornier Magnan B, Serrano J, Bellet H, Saumade R. Activity of citrulline malate on acid-base balance and blood ammonia and amino acid levels. Study in the animal and in man. Arzneimittel-Forschung 41: 660–663, 1991.
16. Campos HO, Drummond LR, Rodrigues QT, et al. Nitrate supplementation improves physical performance specifically in non-athletes during prolonged open-ended tests: A systematic review and meta-analysis. Br J Nutr 119: 636–657, 2018.
17. Chappell AJ, Allwood DM, Johns R, et al. Citrulline malate supplementation does not improve German Volume Training performance or reduce muscle soreness in moderately trained males and females. J Int Soc Sports Nutr 15: 42, 2018.
18. Chappell AJ, Allwood DM, Simper TN. Citrulline Malate fails to improve German volume training performance in healthy young men and women. J Diet Suppl 21: 1–12, 2018.
19. Chen IF, Wu HJ, Chen CY, Chou KM, Chang CK. Branched-chain amino acids, arginine, citrulline alleviate central fatigue after 3 simulated matches in taekwondo athletes: A randomized controlled trial. J Int Soc Sports Nutr 13: 28, 2016.
20. Cheng IS, Wang YW, Chen IF, et al. The supplementation of branched-chain amino acids, arginine, and citrulline improves endurance exercise performance in two consecutive days. J Sport Sci Med 15: 509, 2016.
21. Churchward-Venne TA, Cotie LM, MacDonald MJ, et al. Citrulline does not enhance blood flow, microvascular circulation, or myofibrillar protein synthesis in elderly men at rest or following exercise. Am J Physiol Endocrinol Metab 307: E71–E83, 2014.
22. Coggan AR, Leibowitz JL, Kadkhodayan A, et al. Effect of acute dietary nitrate intake on maximal knee extensor speed and power in healthy men and women. Nitric Oxide 48: 16–21, 2015.
23. Coggan AR, Leibowitz JL, Spearie CA, et al. Acute dietary nitrate intake improves muscle contractile function in patients with heart failure: A double-blind, placebo-controlled, randomized trial. Circ Heart Fail 8: 914–920, 2015.
24. Collins JK, Wu G, Perkins-Veazie P, et al. Watermelon consumption increases plasma arginine concentrations in adults. Nutrition 23: 261–266, 2007.
25. Cooper CE, Giulivi C. Nitric oxide regulation of mitochondrial oxygen consumption II: Molecular mechanism and tissue physiology. Am J Physiol Cell Physiol 292: C1993–C2003, 2007.
26. Cunniffe B, Papageorgiou M, O'Brien B, et al. Acute citrulline-malate supplementation and high-intensity cycling performance. J Strength Cond Res 30: 2638–2647, 2016.
27. Cutrufello PT, Gadomski SJ, Zavorsky GS. The effect of l-citrulline and watermelon juice supplementation on anaerobic and aerobic exercise performance. J Sports Sci 33: 1459–1466, 2015.
28. da Silva DK, Jacinto JL, de Andrade WB, et al. Citrulline malate does not improve muscle recovery after resistance exercise in untrained young adult men. Nutrients 9: 1132, 2017.
29. Drenning JA, Lira VA, Soltow QA, et al. Endothelial nitric oxide synthase is involved in calcium-induced Akt signaling in mouse skeletal muscle. Nitric Oxide 21: 192–200, 2009.
30. El-Bassossy HM, El-Fawal R, Fahmy A. Arginase inhibition alleviates hypertension associated with diabetes: Effect on endothelial dependent relaxation and NO production. Vasc Pharmacol 57: 194–200, 2012.
31. Farney TM, Bliss MV, Hearon CM, Salazar DA. The effect of citrulline malate supplementation on muscle fatigue among healthy participants. J Strength Cond Res 33: 2464–2470, 2019.
32. Fernstrom JD. Branched-chain amino acids and brain function. J Nutr 135: 1539S–1546S, 2005.
33. Figueroa A, Alvarez-Alvarado S, Ormsbee MJ, et al. Impact of L-citrulline supplementation and whole-body vibration training on arterial stiffness and leg muscle function in obese postmenopausal women with high blood pressure. Exp Gerontol 63: 35–40, 2015.
34. Figueroa A, Wong A, Jaime SJ, Gonzales JU. Influence of L-citrulline and watermelon supplementation on vascular function and exercise performance. Curr Opin Clin Nutr Metab Care 20: 92–98, 2017.
35. Flueck JL, Bogdanova A, Mettler S, Perret C. Is beetroot juice more effective than sodium nitrate? The effects of equimolar nitrate dosages of nitrate-rich beetroot juice and sodium nitrate on oxygen consumption during exercise. Appl Physiol Nutr Metab 41: 421–429, 2015.
36. Fornaris E, Vanuxem D, Duflot J. Pharmacoclinical approach of citrulline malate activity: Analysis of blood lactate during a standardised exercise. Gaz Medicale 91: 1–3, 1984.
37. Fouré A, Bendahan D. Is branched-chain amino acids supplementation an efficient nutritional strategy to alleviate skeletal muscle damage? A systematic review. Nutrients 9: 1047, 2017.
38. Glenn JM, Gray M, Jensen A, Stone MS, Vincenzo JL. Acute citrulline-malate supplementation improves maximal strength and anaerobic power in female, masters athletes tennis players. Eur J Sport Sci 16: 1095–1103, 2016.
39. Glenn JM, Gray M, Wethington LN, et al. Acute citrulline malate supplementation improves upper-and lower-body submaximal weightlifting exercise performance in resistance-trained females. Eur J Nutr 56: 775–784, 2017.
40. Gonzales JU, Raymond A, Ashley J, Kim Y. Does l-citrulline supplementation improve exercise blood flow in older adults? Exp Physiol 102: 1661–1671, 2017.
41. Gonzalez AM, Spitz RW, Ghigiarelli JJ, Sell KM, Mangine GT. Acute effect of citrulline malate supplementation on upper-body resistance exercise performance in recreationally resistance-trained men. J Strength Cond Res 32: 3088–3094, 2018.
42. Grimble GK. Adverse gastrointestinal effects of arginine and related amino acids. J Nutr 137: 1693S–1701S, 2007.
43. Harty PS, Zabriskie HA, Erickson JL, et al. Multi-ingredient pre-workout supplements, safety implications, and performance outcomes: A brief review. J Int Soc Sports Nutr 15: 1–28, 2018.
44. Heller R, Unbehaun A, Schellenberg B, et al. L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem 276: 40–47, 2001.
45. Hickner RC, Tanner CJ, Evans CA, et al. L-citrulline reduces time to exhaustion and insulin response to a graded exercise test. Med Sci Sports Exerc 38: 660–666, 2006.
46. Hsueh CF, Wu HJ, Tsai TS, Wu CL, Chang CK. The effect of branched-chain amino acids, citrulline, and arginine on high-intensity interval performance in young swimmers. Nutrients 10: 2018, 1979.
47. Hwang P, Marroquín FEM, Gann J, et al. Eight weeks of resistance training in conjunction with glutathione and L-Citrulline supplementation increases lean mass and has no adverse effects on blood clinical safety markers in resistance-trained males. J Int Soc Sports Nutr 15: 30, 2018.
48. Ignarro LJ, Byrns RE, Sumi D, de Nigris F, Napoli C. Pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide. Nitric Oxide 15: 93–102, 2006.
49. Jagim AR, Harty PS, Camic CL. Common ingredient profiles of multi-ingredient pre-workout supplements. Nutrients 11: 254, 2019.
50. Jones AM. Dietary nitrate supplementation and exercise performance. Sports Med 44: 35–45, 2014.
51. Jourdan M, Nair KS, Carter RE, et al. Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet-A pilot study. Clin Nutr 34: 449–456, 2015.
52. Kerley CP. Dietary nitrate as modulator of physical performance and cardiovascular health. Curr Opin Clin Nutr Metab Care 20: 440–446, 2017.
53. Kim I-Y, Schutzler SE, Schrader A, et al. Acute ingestion of citrulline stimulates nitric oxide synthesis but does not increase blood flow in healthy young and older adults with heart failure. Am J Physiol Endocrinol Metab 309: E915–E924, 2015.
54. Kiyici F, Eroğlu H, Kishali NF, Burmaoglu G. The effect of citrulline/malate on blood lactate levels in intensive exercise. Biochem Genet 55: 387–394, 2017.
55. Le Roux-Mallouf T, Laurent J, Besset D, et al. Effects of acute NO precursor intake on peripheral and central fatigue during knee extensions in healthy men. Exp Physiol 104: 1100–1114, 2019.
56. Le Roux-Mallouf T, Vibert F, Doutreleau S, Verges S. Effect of acute nitrate and citrulline supplementation on muscle microvascular response to ischemia–reperfusion in healthy humans. Appl Physiol Nutr Metab 42: 901–908, 2017.
57. Leiter JR, Upadhaya R, Anderson JE. Nitric oxide and voluntary exercise together promote quadriceps hypertrophy and increase vascular density in female 18-mo-old mice. Am J Physiol Cell Physiol 302: C1306–C1315, 2012.
58. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov 7: 156, 2008.
59. MacLean D, Graham T. Branched-chain amino acid supplementation augments plasma ammonia responses during exercise in humans. J Appl Physiol 74: 2711–2717, 1993.
60. MacLean D, Graham T, Saltin B. Stimulation of muscle ammonia production during exercise following branched-chain amino acid supplementation in humans. J Physiol 493: 909–922, 1996.
61. Mahboobi S, Tsang C, Rezaei S, Jafarnejad S. Effect of l-citrulline supplementation on blood pressure: A systematic review and meta-analysis of randomized controlled trials. J Hum Hypertens 33: 10–21, 2018.
62. Mandel H, Levy N, Izkovitch S, Korman S. Elevated plasma citrulline and arginine due to consumption of Citrullus vulgaris (watermelon). J Inherit Metab Dis 28: 467–472, 2005.
63. Martinez-Sanchez A, Alacid F, Rubio-Arias JA, et al. Consumption of watermelon juice enriched in L-citrulline and pomegranate ellagitannins enhanced metabolism during physical exercise. J Agric Food Chem 65: 4395–4404, 2017.
64. Martínez-Sánchez A, Ramos-Campo DJ, Fernández-Lobato B, et al. Biochemical, physiological, and performance response of a functional watermelon juice enriched in L-citrulline during a half-marathon race. Food Nutr Res 61: 1330098, 2017.
65. McKinley-Barnard S, Andre T, Morita M, Willoughby DS. Combined L-citrulline and glutathione supplementation increases the concentration of markers indicative of nitric oxide synthesis. J Int Soc Sports Nutr 12: 27, 2015.
66. Meneguello M, Mendonca J, Lancha A Jr, Costa Rosa L. Effect of arginine, ornithine and citrulline supplementation upon performance and metabolism of trained rats. Cell Biochem Funct 21: 85–91, 2003.
67. Meyer RA, Dudley GA, Terjung RL. Ammonia and IMP in different skeletal muscle fibers after exercise in rats. J Appl Physiol 49: 1037–1041, 1980.
68. Moinard C, Nicolis I, Neveux N, et al. Dose-ranging effects of citrulline administration on plasma amino acids and hormonal patterns in healthy subjects: The citrudose pharmacokinetic study. Br J Nutr 99: 855–862, 2008.
69. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 329: 2002–2012, 1993.
70. Morita M, Hayashi T, Ochiai M, et al. Oral supplementation with a combination of L-citrulline and L-arginine rapidly increases plasma L-arginine concentration and enhances NO bioavailability. Biochem Biophys Res Commun 454: 53–57, 2014.
71. Morita M, Sakurada M, Watanabe F, et al. Effects of oral L-citrulline supplementation on lipoprotein oxidation and endothelial dysfunction in humans with vasospastic angina. Immunol Endocr Metab Agents Med Chem 13: 214–220, 2013.
72. Mosher SL, Sparks SA, Williams EL, Bentley DJ, McNaughton LR. Ingestion of a nitric oxide enhancing supplement improves resistance exercise performance. J Strength Cond Res 30: 3520–3524, 2016.
73. Mutch B, Banister E. Ammonia metabolism in exercise and fatigue: A review. Med Sci Sports Exerc 15: 41–50, 1983.
74. Ochiai M, Hayashi T, Morita M, et al. Short-term effects of L-citrulline supplementation on arterial stiffness in middle-aged men. Int J Cardiol 155: 257–261, 2012.
75. Papadia C, Osowska S, Cynober L, Forbes A. Citrulline in health and disease. Review on human studies. Clin Nutr 37: 1823–1828, 2018.
76. Pechánová O, Kashiba M, Inoue M. Role of glutathione in stabilization of nitric oxide during hypertension developed by inhibition of nitric oxide synthase in the rat. Jpn J Pharmacol 81: 223–229, 1999.
77. Pereira C, Ferreira NR, Rocha BS, Barbosa RM, Laranjinha J. The redox interplay between nitrite and nitric oxide: From the gut to the brain. Redox Biol 1: 276–284, 2013.
78. Pérez-Guisado J, Jakeman PM. Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness. J Strength Cond Res 24: 1215–1222, 2010.
79. Rimando AM, Perkins-Veazie PM. Determination of citrulline in watermelon rind. J Chromatogr 1078: 196–200, 2005.
80. Roelofs EJ, Smith-Ryan AE, Trexler ET, Hirsch KR, Mock MG. Effects of pomegranate extract on blood flow and vessel diameter after high-intensity exercise in young, healthy adults. Eur J Sport Sci 17: 317–325, 2017.
81. Romero MJ, Platt DH, Caldwell RB, Caldwell RW. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev 24: 275–290, 2006.
82. Rougé C, Des Robert C, Robins A, et al. Manipulation of citrulline availability in humans. Am J Physiol Gastrointest Liver Physiol 293: G1061–G1067, 2007.
83. Schwarz NA, McKinley-Barnard SK. Acute oral ingestion of a multi-ingredient preworkout supplement increases exercise performance and alters postexercise hormone responses: A randomized crossover, double-blinded, placebo-controlled trial. J Diet Suppl 4: 1–16, 2018.
84. Schwedhelm E, Maas R, Freese R, et al. Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: Impact on nitric oxide metabolism. Br J Clin Pharmacol 65: 51–59, 2008.
85. Shanely RA, Nieman DC, Perkins-Veazie P, et al. Comparison of watermelon and carbohydrate beverage on exercise-induced alterations in systemic inflammation, immune dysfunction, and plasma antioxidant capacity. Nutrients 8: 518, 2016.
86. Smith LW, Smith JD, Criswell DS. Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload. J Appl Physiol 92: 2005–2011, 2002.
87. Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol Rev 81: 209–237, 2001.
88. Sureda A, Córdova A, Ferrer MD, et al. L-citrulline-malate influence over branched chain amino acid utilization during exercise. Eur J Appl Physiol 110: 341–351, 2010.
89. Suzuki I, Sakuraba K, Horiike T, et al. A combination of oral l-citrulline and l-arginine improved 10-min full-power cycling test performance in male collegiate soccer players: A randomized crossover trial. Eur J Appl Physiol 119: 1075–1084, 2019.
90. Suzuki T, Morita M, Hayashi T, Kamimura A. The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males. Biosci Biotechnol Biochem 81: 372–375, 2017.
91. Suzuki T, Morita M, Kobayashi Y, Kamimura A. Oral L-citrulline supplementation enhances cycling time trial performance in healthy trained men: Double-blind randomized placebo-controlled 2-way crossover study. J Int Soc Sports Nutr 13: 6, 2016.
92. Takeda K, Machida M, Kohara A, Omi N, Takemasa T. Effects of citrulline supplementation on fatigue and exercise performance in mice. J Nutr Sci Vitamintol 57: 246–250, 2011.
93. Tarazona-Díaz MP, Alacid F, Carrasco M, Martínez I, Aguayo E. Watermelon juice: Potential functional drink for sore muscle relief in athletes. J Agric Food Chem 61: 7522–7528, 2013.
94. Tillin NA, Moudy S, Nourse KM, Tyler CJ. Nitrate supplement benefits contractile forces in fatigued but not unfatigued muscle. Med Sci Sports Exerc 50: 2122–2131, 2018.
95. Totzeck M, Schicho A, Stock P, et al. Nitrite circumvents canonical cGMP signaling to enhance proliferation of myocyte precursor cells. Mol Cell Biochem 401: 175–183, 2015.
96. Trexler ET, Persky AM, Ryan ED, et al. Acute effects of citrulline supplementation on high-intensity strength and power performance: A systematic review and meta-analysis. Sports Med 49: 707–718, 2019.
97. Trexler ET, Smith-Ryan AE, Melvin MN, Roelofs EJ, Wingfield HL. Effects of pomegranate extract on blood flow and running time to exhaustion. Appl Physiol Nutr Metab 39: 1038–1042, 2014.
98. van de Poll MC, Siroen MP, van Leeuwen PA, et al. Interorgan amino acid exchange in humans: Consequences for arginine and citrulline metabolism. Am J Clin Nutr 85: 167–172, 2007.
99. Vanhoutte PM, Zhao Y, Xu A, Leung SW. Thirty years of saying NO: Sources, fate, actions, and misfortunes of the endothelium-derived vasodilator mediator. Circ Res 119: 375–396, 2016.
100. Wax B, Kavazis AN, Luckett W. Effects of supplemental citrulline-malate ingestion on blood lactate, cardiovascular dynamics, and resistance exercise performance in trained males. J Diet Suppl 13: 269–282, 2016.
101. Wax B, Kavazis AN, Weldon K, Sperlak J. Effects of supplemental citrulline malate ingestion during repeated bouts of lower-body exercise in advanced weightlifters. J Strength Cond Res 29: 786–792, 2015.
102. Windmueller HG, Spaeth AE. Source and fate of circulating citrulline. Am J Physiol Endocrinol Metab 241: E473–E480, 1981.
103. Wu G. Urea synthesis in enterocytes of developing pigs. Biochem J 312: 717–723, 1995.
104. Wu G, Morris SM. Arginine metabolism: Nitric oxide and beyond. Biochem J 336: 1–17, 1998.
105. Yang CC, Wu CL, Chen IF, Chang CK. Prevention of perceptual‐motor decline by branched-chain amino acids, arginine, citrulline after tennis match. Scand J Med Sci Spor 27: 935–944, 2017.
