Background
NO plays key roles such as maintaining the function and integrity of the endothelium, including vascular tone and structure [
1]. In sports physiology, nitrate supplementation is thought to be an ergogenic aid [
2‐
4]. This view is based on evidence that NO is an important modulator of blood flow and mitochondrial respiration under physiological conditions [
5]. Some studies have shown that dietary NO related supplements, such as nitrate-rich beetroot juice, enhance human sport performance [
6‐
9]. Dietary supplementation with nitrate thus appears to be beneficial for exercise.
There is growing interest in the use of L-citrulline as an NO-related dietary ingredient. L-Citrulline is present in the body and is a potent endogenous precursor of L-arginine [
10], which is a substrate for NO synthase (NOS). NOS catalyzes a complex enzymatic reaction that leads to NO formation from L-arginine and oxygen and generates L-citrulline as a byproduct [
11]. L-Citrulline is effectively recycled via the L-citrulline NO cycle to L-arginine and plays an important role in the metabolism and regulation of NO [
12]. L-Citrulline supplementation has various beneficial effects, such as ameliorating arterial stiffness [
13] and improving erectile function [
14], memory [
15], O
2 uptake kinetics, and high-intensity exercise performance [
16] through upregulation of NO synthesis. We and others have demonstrated in animal models that oral supplementation with L-citrulline upregulates endothelial NO synthase (eNOS) expression, improves endothelial function, and plays an atheroprotective role [
17‐
19]. Interestingly, a clinical trial has shown that oral intake of L-citrulline dose-dependently and more effectively increases plasma L-arginine levels than does L-arginine supplementation in healthy human volunteers [
20]. Therefore, L-citrulline may be considered an effective L-arginine and NO supplies which might be expected to have potential for enhancing sport performance. Some studies have found that acute L-citrulline supplementation has no effect on exercise [
21,
22]. On the other hand, Bailey et al. [
16] showed that 6 days of L-citrulline supplementation improved exercise tolerance. This suggests that chronic L-citrulline supplementation (for about 1 week) is needed to enhance exercise tolerance. However, it is not presently known whether chronic small doses of L-citrulline enhance sport performance. Moreover, to our knowledge, no study has comprehensively evaluated the effects of L-citrulline on endurance exercise performance during simulated competition or on subjective feelings of discomfort associated with exercise in humans. We hypothesized that chronic L-citrulline administration would enhance performance during simulated competition.
The aim of this study was to investigate the effect of oral supplementation of L-citrulline on cycling time-trial performance in healthy trained men.
Discussion
We demonstrated that oral supplementation with L-citrulline at 2.4 g/day for 7 days significantly increased plasma L-arginine levels. Moreover, intake of L-citrulline for 7 days and I h before the TT significantly increased plasma L-citrulline and L-arginine levels and enhanced cycling TT performance. In addition, subjective feelings of muscle fatigue, and concentration, right after exercise were significantly improved with L-citrulline.
In this human trial, the subjects engaged in TT cycling to allow us to evaluate their exercise performance. Competitive sports typically require athletes to complete a given distance in the shortest possible time. Time-to-exhaustion tests are primarily measures of “exercise capacity,” and because there is no competitive sports event in which competition is based on time and distance before exhaustion, tests of this type have limited physiological validity [
36]. It has also been reported that there is no relationship between measured time-to-exhaustion and actual cycling performance [
37]. In contrast, the TT protocol used here has a high level of physiological validity [
36], provides an accurate simulation of physiological responses during competition [
38], and is well correlated with actual race performance [
39]. Therefore, L-citrulline intake might be expected to enhance performance in real competitive sport. We used cornstarch as the placebo and the study design was a double-blind crossover; we expected that these factors would reduce the placebo effect.
The improved TT performance after supplementation with L-citrulline was the consequence of significantly greater PO for the same VO
2. Interestingly, there was a significant correlation between plasma NOx and PO/VO
2 after supplementation with L-citrulline but placebo had no correlation (data not shown). This finding suggests that the effects of L-citrulline on PO/VO
2 may have been related to improved plasma NO availability, which in turn may have enhanced sport performance. In sports nutrition, NO-related products are attracting a lot of attention for their ergogenic effects. Many applied studies in humans report that NO improves sports performance. This is because NO is a potential modulator of blood flow, muscle energy metabolism, and mitochondrial respiration during exercise [
5,
6,
40,
41]. Dietary supplementation with nitrate reduces the O
2 cost of submaximal cycling [
6,
42], knee extensor exercise [
43], and treadmill walking and running [
44]. L-Arginine is the direct precursor of NO via NOS activity. Moreover, oral intake of L-arginine improves sports performance in healthy subjects [
45‐
47]. However, a relatively large dose (6 to 14.2 g/day) of L-arginine would be required for beneficial effects on sports performance, because L-arginine is degraded by arginase in the small intestine and liver [
48‐
50]. For this reason, some studies have shown no effect of L-arginine on O
2 cost and sports performance [
51,
52]. In contrast, L-citrulline is not metabolized in the intestine or liver [
53]. On entering the kidneys, vascular endothelium, and other tissues, L-citrulline is readily converted to L-arginine, thus raising plasma and tissue levels of L-arginine and enhancing NO production [
10]. We found here that oral intake of L-citrulline increased not only L-citrulline levels but also L-arginine levels. It has been reported that oral supplementation with L-citrulline increases plasma L-arginine levels more effectively than does L-arginine supplementation in healthy subjects [
20], and increased plasma L-arginine levels before exercise enhance sport performance [
16]. However, some studies have found that oral L-citrulline supplementation has no effect on exercise [
21,
22]. This is likely because a single dose of L-citrulline is insufficient to enhance sport performance. Bailey et al. [
16] demonstrated that 6 days of L-citrulline supplementation improved exercise tolerance. These findings suggest that L-citrulline needs to be taken continuously (for about 1 week) to enhance exercise tolerance. This is why we conducted an 8-day trial, which showed positive effects of L-citrulline. The daily dose of L-citrulline in our study was 2.4 g. This seems smaller than those used in other previous studies [
16,
21,
22], but 2 to 3 g of oral L-citrulline has been reported to increase plasma L-arginine levels [
20,
54]. Moinard et al. [
54] showed that the C
max of plasma L-arginine was 146 μM when subjects consumed 2 g of L-citrulline. In the study by Bailey et al. [
16], the L-citrulline group had a mean plasma L-arginine level of 135 μM and showed improved exercise performance. Therefore, we had hypothesized that 2.4 g/day of L-citrulline for 8 days might be enough to increase plasma L-arginine levels such that we would obtain ergogenic effects. In fact, Bailey et al. reported that the plasma L-arginine level in their L-citrulline group was about 2.3 times that in their placebo group [
16]. On the other hand, our data demonstrated that the plasma L-arginine level in our L-citrulline group was about 1.7 times that in our placebo group. However, our absolute value of plasma L-arginine after intake of L-citrulline was higher than that in the study by Bailey et al. Therefore, 2.4 g/day of L-citrulline for 8 days is likely enough to obtain ergogenic aid.
We found here that, L-citrulline supplementation significantly increased plasma levels of plasma L-citrulline and L-arginine, which are essential for NO synthesis. In our study the plasma arginine level after supplementation of L-citrulline was as high as that in the study by Bailey et al. [
16]. Our results thus suggest that L-citrulline would enhance sport performance through NO synthesis; however, we were not able to observe an increase in plasma NOx level. We measured NOx at only two time points: before and after exercise, not during exercise. These evaluation points might not have been suitable for detecting significant between-point differences in NO generation. Chemiluminescence assay is more sensitive than colorimetric assay for detecting NOx. In this study, we measured NOx by colorimetric assay, and it may not have been sensitive enough to detect changes in plasma NOx.
Sureda et al. [
26] showed that oral intake of 6 g of L-citrulline malate 2 h before exercise enhances the use of BCAAs, which are metabolized in the muscles to produce energy. In our study, L-citrulline supplementation decreased plasma BCAA levels. These data indicate that L-citrulline promotes the metabolic use of these amino acids as fuel to support muscular exercise. Moreover, L-citrulline significantly improved subjective feelings of muscular fatigue. BCAA reduces muscle soreness and fatigue [
55,
56]. Furthermore, L-citrulline malate reduces fatigue and post-workout muscle soreness [
25]; watermelon juice, which is rich in L-citrulline, also reduces muscle soreness [
57]. Our data suggest that L-citrulline has the potential to relieve muscle fatigue. Therefore, the effects of L-citrulline on BCAA utilization and muscular fatigue might also contribute to enhanced sport performance. In addition, the subjective feeling of concentration was significantly improved by oral intake of L-citrulline. Hayashi et al. have reported that L-citrulline improves blood flow [
17]. The concentration-enhancing effects of L-citrulline are likely due to enhanced blood flow.
A growing number of sport supplements include L-arginine, which is claimed to enhance NO production, despite L-arginine being rapidly metabolized in the small intestine and liver when administered orally. As mentioned above, L-citrulline is a potent precursor of L-arginine, and several functional advantages of L-citrulline over L-arginine have been elucidated [
17,
20]; moreover, Akashi et al. [
58] have revealed that L-citrulline is an efficient hydroxyl radical scavenger. L-arginine tends to be extremely bitter and highly water absorbent, whereas L-citrulline is tasteless, odorless, and non-hygroscopic. It would thus appear that L-citrulline is superior to L-arginine in terms of ease of handling and taste as an ingredient of supplements. L-Citrulline is present in large quantities in watermelon but is not abundant in other fruits, vegetables, meat, or fish because it is a free amino acid. It is difficult to obtain L-citrulline from a conventional diet in sufficient amounts to enhance sports performance. Therefore, it may be beneficial to take a few tablets of L-citrulline before exercise as an ergogenic aid.
Our study had several limitations. We instructed the subjects not to change their training volumes and to eat their usual diets during the 7 days of the study. However, we did not make the training volumes and diets identical among the subjects, with the exception of dinner on the evening before the test day and breakfast on the test day. Some of the subjects’ performances may have been affected by intense training sessions in the 2 days before the trials. Here, we conducted a double-blind randomized placebo-controlled two-way crossover study in 22 subjects; in future, an additional, larger-scale study will be needed to verify our findings.
Competing interests
This study was conducted in research funding by KYOWA HAKKO BIO CO., LTD. Takashi Suzuki, Masahiko Morita and Ayako Kamimura are employee of KYOWA HAKKO BIO CO., LTD. The other co-author declare no conflict of interests.
Authors’ contributions
TS carried out study design and drafted the manuscript. MM made contributions to design, helped to draft the manuscript. YK made contributions to conception and design. AK supervised manuscript preparation. All authors read and approved the final manuscript.