Potential role of L-citrulline in regulating exercise performance and muscle protein metabolism

Abstract

L-citrulline (L-Cit) has emerged as a potential supplement to enhance muscle performance and protein metabolism. This review summarizes evidence from rodent and human studies, highlighting its effects on muscle function, protein synthesis, and underlying mechanisms. Key areas for future research include supplementation strategies, transport and metabolism pathways, mitochondrial function, and the interaction between L-Cit, gut microbiota, and muscle health, offering insights for nutritional interventions targeting aging and sarcopenia.

Introduction

The regulation of muscle performance and protein turnover is a key focus in health research. Skeletal muscle mass undergoes significant postnatal growth, reaching its peak in adulthood¹. As the body’s primary protein reservoir, skeletal muscle accounts for 50–60% of total adult protein content, with proteins comprising 88% of muscle weight in a dehydrated state. Muscle tissue exhibits high plasticity, with its mass varying in response to nutritional status. The maintenance of muscle mass relies on precisely controlled protein synthesis and breakdown². Muscle growth occurs when protein synthesis exceeds degradation, whereas muscle loss ensues when degradation surpasses synthesis. This equilibrium between synthesis and degradation is crucial for maintaining health across the lifespan.

The decline in muscle mass in the human body is associated with diminished athletic performance and heightened susceptibility to falls and fractures, thereby compromising individuals’ ability to maintain a normal lifestyle. This reduction in muscle mass is an inevitable consequence of aging, indicative of declining physical function. Muscle mass typically begins to decrease around the age of 40, with a notable acceleration in loss by the age of 70, a phenomenon commonly referred to as sarcopenia³. Recognizing its profound clinical impact, the World Health Organization (WHO) officially classified sarcopenia as a unique disease in 2015 and named it ICD-10 disease code⁴. Furthermore, many recent meta-analyses have highlighted the serious risks associated with this situation. These studies indicate that sarcopenia significantly increases the likelihood of adverse health outcomes, including higher all-cause mortality, functional decline, postoperative complications, and prolonged hospital stays^5–8. In low-income countries, the high prevalence of sarcopenia poses a significant societal and healthcare burden⁹. Thus, preserving muscle mass and optimal athletic performance is crucial for promoting human health and enhancing the quality of life, particularly among older adults. The maintenance of muscle mass and physical performance is crucial for the health and quality of life of the elderly population. To combat sarcopenia, people have explored various nutritional and regulatory strategies. The current countermeasures include a series of dietary intervention measures, such as a high-protein diet, pulse-feeding patterns, strategic intake of slow-digest and fast-digest proteins, and vitamin D supplements^10–12. Among these different methods, targeted amino acid supplementation has become an effective strategy. Among the amino acids, branched-chain amino acids (BCAAs) have been extensively studied and are currently the most effective for stimulating muscle hypertrophy. However, in order to overcome muscle atrophy and optimize treatment strategies for vulnerable groups, new alternative options are being actively studied. Although L-citrulline (L-Cit) was first isolated and identified in the early 20th century¹³, its crucial role in regulating muscle metabolism has only been clarified recently. Recently, it has been discovered that it can stimulate muscle protein synthesis and increase muscle mass, providing a new nutritional strategy for improving physical function^14–16. Aging has been associated with increased sequestration of amino acids by the spleen and the development of sarcopenia¹⁷. In aging populations, the phenomenon of “anabolic resistance”—a blunted Muscle protein synthesis (MPS) response to dietary amino acids—accelerates the onset of sarcopenia¹⁸. While BCAAs, particularly leucine, are well-established triggers for MPS via the mTOR pathway, their efficacy can be limited by splanchnic extraction¹⁹. Herein lies the unique metabolic advantage of L-Cit. L-Cit is transported from the enterocyte directly to the portal circulation, bypassing the liver¹⁵. It is subsequently converted to arginine in the kidneys, effectively increasing systemic arginine bioavailability more efficiently than arginine supplementation itself. Therefore, the main purpose of this review is to evaluate the efficacy of L-Cit in reducing muscle loss and improving exercise, and to assess the differences between preclinical mechanisms and clinical outcomes. Furthermore, this review aims to clarify the effects and regulatory pathways of L-Cit on muscle protein synthesis, providing insights into its therapeutic potential in improving muscle health in the elderly population.

Methodology

Literature retrieval strategy: Systematic searches were conducted using PubMed, Web of Science, and Scopus (up to December 2025). The keywords include “L-citrulline”, “citrulline malate”, “muscle protein synthesis”, “mTOR”, “sarcopenia” and “athletic performance”. Inclusion criteria prioritized randomized controlled trials (RCTs) in humans and mechanistic animal studies published within the last 15 years. Studies using multi-ingredient blends (where L-Cit effects could not be isolated) were excluded or explicitly noted as confounding.

Biochemical properties of L-Cit

Discovery of L-Cit

L-Cit was first isolated from watermelon juice by Yotaro Koga and Ryo Ohtake¹³. 110 years ago, but it was only in 1930 that it was confirmed as an amino acid²⁰. Classified as a non-essential amino acid, L-Cit is synthesized by the human body²¹. Unlike the other 20 amino acids, L-Cit does not directly contribute to protein synthesis, although it can form peptide bonds²². Watermelon (Citrullus lanatus) is the primary natural source of L-Cit, with cucumber, pumpkin, melon, bitter gourd, and gourd also containing it. L-Cit can be extracted from various fruits in the Cucurbitaceae family^23–25. In watermelon, L-citrulline is found in both the flesh and peel, with higher concentrations in the peel²⁶. From an industrial perspective, L-Cit is primarily produced through microbial fermentation and bioconversion processes, such as bioconversion from corn starch or enzymatic production from arginine in fermenters^27,28. While industrial manufacturing relies on these bioprocesses, watermelon remains the most abundant natural dietary source of L-Cit. Typically, watermelon contains 0.7–3.6 g/kg of L-citrulline per fresh weight²⁶, while the peel contains 0.18–2.41 mg/g, varying by variety^25,29.

Transfer of L-Cit

The transport mechanisms of L-citrulline have been well-documented in various cell types, including nerve³⁰, renal tubular³¹, macrophage³², aortic endothelial³³, and glial cells. While foundational studies, such as the work by Bahri et al.³⁴, have evaluated intestinal L-Cit uptake in intestinal epithelial cell models, the comprehensive in vivo mechanisms of its intestinal transport have received comparatively less attention. Previously, the intestinal transport of L-citrulline was considered insignificant due to its lack of direct involvement in protein synthesis. Despite L-citrulline’s role in enhancing skeletal muscle mass and reducing sarcopenia, the primary source of amino acids for muscle protein synthesis is the absorption from intestinal chyme. Although L-Cit has been shown to enhance skeletal muscle mass³⁵ and decrease sarcopenia³⁶, the majority of amino acids essential for muscle protein synthesis are sourced from the absorption of amino acids in chyme within the intestines. While L-Cit itself does not directly engage in skeletal muscle protein synthesis, it could potentially influence the uptake and absorption of other amino acids within the intestinal lumen. This review seeks to clarify the transport mechanisms of L-Cit in intestinal epithelial and skeletal muscle cells.

While the precise molecular mechanisms of L-Cit transport remain to be fully defined, numerous in vivo studies have unequivocally demonstrated its high bioavailability and efficient absorption in both animals and humans^37,38. Structurally, L-Cit resembles substrates for several transporters. Putative candidates in the intestine include BAT1 (SLC6A19), the b^[0,+]AT1-rBAT complex, LAT2 (SLC7A8), and y + LAT^31,39–42. Although reduced L-Cit levels in LAT4 knockout mice imply a potential role for LAT4 in L-Cit exchange^43,44, direct empirical evidence for these specific transporters in L-Cit uptake remains limited.

Importantly, the physiological relevance of L-citrulline transport depends on its distinct inter-organ metabolism, which will be discussed in detail in “Metabolism of L-Cit”. Unlike many other amino acids, L-Cit largely bypasses hepatic first-pass metabolism. Its efficient intestinal absorption allows it to reach the systemic circulation and subsequently enter skeletal muscle. In myocytes, while the specific uptake mechanisms (potentially involving SNATs or CATs) are still being elucidated, the transported L-Cit plays a vital metabolic role. It is an efficient precursor for intracellular arginine synthesis, thereby stimulating the production of nitric oxide (NO) and the mTOR signaling pathway - these pathways are crucial for muscle protein synthesis and the fight against sarcopenia.

Given the substrate-specific overlap and unclear subcellular localization of these transport proteins, the analysis of L-Cit transport activity remains very difficult. Recent approaches, such as predicting membrane transport proteins using deep learning embeddings⁴⁵, offer new avenues. However, direct in vivo evidence of L-Cit transport rates in human muscle tissue is currently limited. Future research must validate these putative mechanisms using stable isotope tracer studies and utilize computer modeling to simulate transient L-Cit fluxes based on plasma L-Cit fluctuations and mRNA expression data, particularly in sarcopenia-affected muscle models⁴⁶ (Fig. 1).

Fig. 1. Potential transporters of citrulline in the intestine and muscle include B0AT1 and B0, +AT1.

Additionally, LAT4 may facilitate the exchange of L-citrulline through its coupling role with LAT2/y + LAT1. Cit, citrulline; Leu, leucine; Ile, isoleucine; Val, valine; SLC6A19 (B0AT1), solute carrier family 6 member 19; SLC7A9 (b0,+AT1), solute carrier family 7 member 9; LAT2, large neutral amino acid transporter 2; LAT4, large neutral amino acid transporter 4; SLC7A7 (y + LAT1), solute carrier family 7 member 7. Illustration created with BioRender.com.

Metabolism of L-Cit

The metabolic pathway of L-Cit sets it apart from other amino acids. Following the consumption of arginine (ARG) or glutamine (GLN), the small intestine notably releases a significant quantity of L-Cit, with the small intestine being the exclusive origin of circulating L-Cit⁴⁷. L-Cit represents the end product of intestinal glutamine metabolism, contributing to 27.6% of the nitrogen metabolized from glutamine⁴⁷. Within the intestine, ARG can generate L-Cit via the actions of arginase II and ornithine carbamoyl transferase (Fig. 2). The flux of this intestinal-renal axis is dynamically regulated by dietary protein intake to maintain systemic nitrogen homeostasis. Research has shown a close positive relationship between protein supply and the portal flow of both ARG and L-Cit, indicating that citrulline synthesis scales with nitrogen availability⁴⁸. Interestingly, under a low-protein diet, the expression of OTC in the intestine was significantly enhanced⁴⁹, which might be a compensatory mechanism that maximizes the production of L-Cit in the presence of nitrogen deficiency to serve the supply of ARG. Furthermore, the expression of other key enzymes in the intestinal ARG-ornithine L-Cit is sensitive to protein supply levels⁵⁰, highlighting the role of L-Cit as a key intermediate for adapting to different nutritional states. Nevertheless, L-Cit remains intact in the intestinal tract due to the limited enzymatic activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), which are crucial for its breakdown⁵¹. Furthermore, although the liver has a complete enzymatic mechanism for the urea cycle, L-Cit in the cycle is not metabolized by the liver. As Deutz et al.⁵² demonstrated, the liver lacks the transport mechanism required for the uptake of L-Cit from the systemic circulation. This liver bypass highlights its unique renal uptake, accounting for approximately 75% of circulating L-cysteine extraction (Fig. 2). In the kidneys, it shows high ASS and ASL activity but lacks arginase, and the extracted L-Cit is effectively converted into arginine. Because the kidneys cannot further degrade this newly synthesized arginine, it leads to the net release of ARG into the systemic circulation. Therefore, the intestinal synthesis rate of L-Cit is the main driver of renal ARG production⁵³, and the plasma L-Cit level indicates the balance between intestinal synthesis and renal conversion to ARG.

Fig. 2. Interorgan metabolism of L-Cit and its role in Arg homeostasis.

This interorgan flux is sensitive to nutritional status: dietary protein supply positively correlates with the portal flow of L-Cit, while low-protein intake induces intestinal OTC expression to prioritize nitrogen conservation. The small intestine exclusively synthesizes circulating L-Cit via glutamine (GLN) metabolism (27.6% of GLN-derived nitrogen) or ARG conversion by arginase II and ornithine carbamoyl transferase (OCT). L-Cit escapes intestinal/hepatic degradation due to low argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) activity in the gut and negligible hepatic uptake. Instead, 75% of circulating L-Cit is absorbed by the kidneys, where ASS and ASL convert it to ARG. The production of ARG in the kidneys depends on the synthesis of L-Cit in the intestine. The ARG-CIT-ARG cycle protects ARG from degradation by arginase in the liver. During the period of ARG deficiency, supplementing L-Cit bypasses liver metabolism, accumulates in peripheral tissues (such as muscles), and supports protein synthesis without changing the L-Cit level in the liver. This tissue-specific accumulation may ameliorate sarcopenia, as muscles lack ASS/ASL and thus retain L-Cit. Cit, citrulline; Arg, arginine. Illustration created with BioRender.com.

The liver degrades Arg primarily via arginase; however, the Arg-Cit-Arg interorgan circulation protects Arg from excessive hepatic degradation. When protein intake is insufficient, L-Cit supplementation serves as an efficient precursor to support protein homeostasis. Unlike Arg, L-Cit bypasses hepatic sequestration because the liver lacks significant citrulline uptake capacity, allowing L-Cit to reach the systemic circulation intact¹⁶ (Fig. 2). This “bypass” effect results in a more sustained increase in plasma Arg levels than oral Arg supplementation itself. Importantly, L-Cit directly stimulates muscle protein synthesis in myotubes that do not quantitatively express argininosuccinate synthase nor argininosuccinate lyase (the two enzymes required for the transformation of CIT into ARG)⁵¹. Consequently, L-Cit supplementation represents a targeted nutritional strategy to enhance anabolism and potentially alleviate sarcopenia in protein-malnourished or aging populations.

The beneficial effects of Cit on muscles: open to debate

The effect of Cit supplementation on muscle exercise performance and protein synthesis in rodents

In vitro studies on mouse muscle cells revealed that L-Cit reallocates ATP to enhance protein synthesis⁵⁴. Numerous studies in mice have shown L-Cit to enhance exercise performance and increase skeletal muscle mass by promoting protein synthesis^55,56. For example, administering 250 mg/kg of L-Cit to mice post-exercise reduced muscle fatigue and improved performance⁵⁵. Doses of 250, 500, and 1000 mg/kg also effectively reduced muscle damage from acute eccentric exercise⁵⁷. Additionally, L-Cit protected against mitochondrial dysfunction and heat-induced muscle damage in mice⁵⁸. In a mouse model of amyotrophic lateral sclerosis, changes in L-Cit levels and LAT1 transporter activity were observed⁵⁹, suggesting a possible role in mitigating the disease. However, L-Cit did not alleviate muscle atrophy in mice with limb casts, likely due to the lack of exercise or resistance training⁶⁰. Thus, L-Cit’s effectiveness may depend on physical activity intensity. Goron et al. demonstrated the synergistic effect of L-Cit supplementation and endurance training in rats. This enhancement may be related to the ability of L-Cit to regulate energy metabolism, especially by activating the pathway that produces acetyl-CoA⁶¹.

After an 18-hour fast, muscle protein synthesis in rats decreased by 61%, but oral L-Cit administration restored synthesis rates¹⁴. Similarly, L-Cit supplementation increased myofibrillar protein levels in rats with restricted feeding (60% of ad libitum intake)⁶². Faure et al. developed a protein-energy malnourished elderly rat model, showing that an L-Cit-enriched diet significantly improved muscle mass and exercise capacity⁶³. L-Cit has been demonstrated to enhance muscle protein metabolism and content in malnourished elderly rats¹⁵. Additionally, L-Cit increased protein synthesis by 15% in fetuses with intrauterine growth restriction⁶⁴. Initial studies focused on malnourished rats, but subsequent research has investigated L-Cit effects under normal physiological conditions.

In healthy elderly rats, a 3-month regimen of continuous L-Cit supplementation increased muscle mass by 25%, attributed to elevated muscle protein levels and larger muscle fibers¹⁹. This suggests L-Cit may help mitigate aging effects. Similarly, in healthy rat models, the same supplementation led to a 25% muscle mass increase via enhanced protein synthesis. Further studies in healthy rats showed L-Cit boosts muscle strength, reduces energy expenditure during contractions⁶⁵, regulates muscle protein and energy metabolism, improves exercise performance, and stimulates protein synthesis⁶¹. Recent findings indicate that oral L-Cit administration enhances nutrient delivery to rat skeletal muscle, potentially aiding exercise performance⁶⁶.

The effect of L-Cit supplementation on human exercise performance and protein synthesis

In rodent models of aging and malnutrition, L-Cit supplementation has consistently demonstrated the ability to stimulate MPS and increase muscle mass, likely via the phosphorylation of the S6 kinase (S6K1) in the mTORC1 pathway⁶⁷. However, human data remains heterogeneous. Jourdan et al. first demonstrated that L-Cit enhances muscle protein synthesis in humans, showing a 25% increase in synthesis among healthy volunteers after three days of a low-protein diet supplemented with L-Cit, compared to an isonitrogenous mixture of non-essential amino acids³⁵. Despite this, skepticism about L-Cit’s efficacy remains. Although rodent studies indicate that L-Cit supplementation reduces post-exercise lactate levels, suggesting improved aerobic utilization, this effect has not been conclusively observed in humans⁶⁸. Extrapolating rodent results to humans is often inappropriate. Several RCTs have failed to show a significant increase in fractional synthetic rate (FSR) of muscle protein following acute L-Cit ingestion in older adults. Some randomized controlled trials failed to show a significant increase in muscle protein FSR after acute intake of L-Cit in healthy elderly people. Firstly, from the perspective of pharmacokinetics, L-Cit is absorbed rapidly and has a high bioavailability. Unlike L-ARG, L-Cit escapes first-pass metabolism in the liver and shows superior gastrointestinal tolerance because it does not induce nitric oxide-mediated secretory diarrhea, which is usually associated with high-dose arginine supplementation⁶⁹. In rodents^70–72 and humans^37,38,73 consistently indicated that oral L-Cit effectively and rapidly increased systemic L-Cit and arginine levels. It is notable that the urinary loss of L-Cit remains very small (<5%), indicating effective renal protection^37,38. Therefore, the lack of efficacy in some human trials cannot be attributed to intestinal malabsorption or insufficient bioavailability.

Secondly, and more importantly, recent evidence has established a new paradigm, demonstrating that the efficacy of L-Cit is governed by thermodynamic mechanisms related to cellular energy states⁷⁴. MPS is a highly energy-dependent process. In a healthy and energetic state with a high ATP/ADP ratio in cells, the Gibbs free energy released by ATP hydrolysis is fully sufficient to drive MPS, thereby making the supplemented L-Cit redundant. However, under restrictive conditions (for example, amino acid deprivation or energy stress), the cellular energy state is reduced, and MPS independent of classical mTOR signaling is directly halted⁷⁴. Under these specific pressure conditions, L-Cit does not increase the total ATP production or restore the ATP/ADP ratio; Instead, it specifically redistributes mitochondrial ATP flux to the protein synthesis mechanism⁵⁴. Because the global ATP/ADP ratio remains unchanged, this redistribution is achieved by reducing the activation energy of the ATP-consuming reaction required for protein assembly. This thermodynamic mechanism explains why L-Cit is not an effective supplement in healthy individuals but is highly effective under conditions of energy deficiency or physiological stress.

The method of L-Cit supplementation affects muscle performance during exercise

Current L-Cit supplementation methods include L-Cit alone, citrulline malate, and combinations with Arg, BCAAs, and nitrate. Most pre-workout supplements blend caffeine, creatine, β-alanine, amino acids, and nitric oxide (NO) precursors, with about 71% containing L-Cit⁷⁵. This complexity obscures L-Cit’s specific effects, complicating the determination of whether its impact is independent or synergistic. This section explores potential synergies between L-Cit and other supplements.

L-Cit + BCAAs

The combination of taking BCAAs, Arg, and L-Cit has been proven to improve athletic performance in various situations (Table 1). For example, an acute preparation containing 0.085 g/kg of BCAAs, 0.05 g/kg of Arg, and 0.05 g/kg of L-Cit before the swimming test improved high-intensity performance⁷⁶. Similarly, it has been reported that compound supplements of BCAAs, L-Cit and other compounds (such as α-glycerophosphocholine or Arg) can improve cycling performance⁷⁷ and enhance runners’ endurance⁷⁸. Because these studies utilized multi-ingredient cocktails, the observed benefits must be regarded as the overall effect of the formulation rather than attributing the main efficacy to a specific ingredient. Furthermore, although combined supplementation seems to be beneficial for athletic performance or muscle preservation in healthy or elderly populations (for example, supplementing 0.8 grams of L-Cit and 1.6 grams of leucine 57 twice daily for 20 weeks⁷⁹), the metabolic interaction between L-Cit and BCAAs—particularly leucine—may be complex and context-dependent. Notably, Ventura et al. (2013) demonstrated in a 2-week dietary-restricted rat model that while L-Cit alone successfully restored muscle protein synthesis (MPS) and maximum tetanic force, the addition of leucine surprisingly antagonized these beneficial effects of L-Cit⁶². This indicates that the physiological state of the organism determines the synergistic or antagonistic properties of these amino acids. During sports activities, L-Cit supplementation has been proven to increase the utilization rate of BCAAs⁸⁰. Given that muscle protein synthesis is a highly energy-dependent process⁸¹, and recent studies have shown that L-Cit can optimize mitochondrial function in specific cell types, such as T cells during sepsis⁸², studying how the combined mixture of L-Cit and BCAAs globally regulates mitochondrial efficiency, muscle integrity and protein synthesis remains a key area for further rigorous research.

Table 1.

The studies concerning L-Cit supplementation on muscle performance and MPS

L-Cit supplement route	Age	The supplementation period	Professional training or not	Enhance athletic performance?	Improve MPS?	Reference
L-Cit + BCAAs	15.6 years	Short term (before swimming test)	Yes	Yes	Not detected	76
L-Cit + BCAAs	43.7 ± 8.5 years	7-day	Yes	Yes	Not detected	77
L-Cit + BCAAs+
Arg	21.65 + 1.7 years	Short term (60 minutes)	Yes	Yes	Not detected	78
L-Cit + Leu	22 weeks old rats	2 weeks	No	Improved muscle strength with L-Cit alone; antagonized by L-Cit + Leucine	Yes (with L-Cit alone); No (Leucine antagonized L-Cit effects)	62
L-Cit + Leu	65–80 years	20 weeks	No	Yes	Not detected	79
L-Cit-malate	22.3 ± 0.9 years	Short term (2 h)	Yes	Yes	Yes	80
L-Cit +Arg	20–49 years	1 week	No	Not detected	Not detected	87
L-Cit +Arg	18–25 years	6 days	Yes	Yes	Not detected	88
L-Cit + BCAAs+
Arg	20.0 ± 0.8 years	Short term (Before the competition)	Yes	Yes	Not detected	89
Citrulline malate	23 ± 3 years	Short term (1 h before the experiment)	No	Yes	Not detected	91
Citrulline malate	23 ± 3 years	Short term (1 h before the experiment)	No	Yes	Not detected	92
Citrulline malate	24.1 ± 3.9 years	Short term (1 h before the experiment)	No	No	Not detected	157
Citrulline malate	23.5 ± 3.7 years	Short term (1 h before the experiment)	No	No	Not detected	94
L-Cit + Nitrate	60-71 years	1 month	No	Yes	Not detected	158
L-Cit + Nitrate	34.37 ± 7.08 years	9 weeks	Yes	No	Not detected	98
Juice rich in L-Cit	35.3 ± 11.4 years	Short term (2 h before the competition)	No	Yes	Not detected	101
L-Cit +glutathione	22.2 ± 2.4 years	1 week	No	Yes	Not detected	102
L-Cit	25.9 ± 2.0 years	3 days	No	Not detected	Yes	35
L-Cit+whey	65–80 years	Short term (before the experiment)	No	Not detected	No	105
L-Cit	80–92 years	3 weeks	No	Not detected	No (No significant increase in whole-body protein synthesis)	106

L-Cit + Arg

Research on the combined effects of L-Cit and Arg on muscle function and quality is limited. Rodent studies suggest L-Cit can enhance muscle protein synthesis^67,83, potentially involving nitric oxide (NO)-mediated signaling⁸⁴, or thermokinetic energy reallocation under physiological stress. Crucially, this stress-dependent anabolic effect of L-Cit has been translated to humans; a clinical study demonstrated that L-Cit ingestion effectively enhances mixed MPS in healthy adults subjected to a short-term low-protein diet³⁵.

In contrast, several in vitro and in vivo studies have established that Arg does not share this direct anabolic capacity. Notably, Claeyssens et al. (2007) explicitly demonstrated that enteral Arg administration fails to stimulate whole-body or mucosal protein synthesis in healthy human subjects⁸⁵. Despite Arg lacking direct muscle anabolic effects on its own, pharmacokinetic synergy between these two amino acids has been clearly demonstrated in humans⁸⁶. For instance, a clinical study demonstrated that combined oral supplementation of L-Cit and Arg (1 gram each) was more effective in increasing plasma L-arginine levels than using an equivalent dose (2 grams) of either amino acid alone⁸⁷. This synergistic enhancement of systemic arginine availability is proposed to optimize the synthesis of endothelial nitric oxide (NO), thereby promoting vasodilation and improving endurance performance⁸⁷ (Table 1). Daily intake of 1.2 g of each supplement for a week has been associated with improved athletic performance in soccer players⁸⁸ (Table 1). Additionally, the concurrent use of L-Cit and Arg may reduce central fatigue in taekwondo athletes⁸⁹ (Table 1). In healthy men, a daily combination of 1 g Arg and 1g L-Cit is more effective at increasing plasma Arg levels than separate 2 g doses of each supplement⁸⁷. While L-Cit is indicated to support muscle protein synthesis, further investigation is required to determine Arg’s role in this process. It is currently unclear whether the combination of L-Cit and Arg can synergistically promote protein synthesis. In the future, active exploration should be conducted on the synergistic effects of L-Cit and Arg on muscle performance, quality, and protein synthesis to determine the effects of separate and mixed supplementation of L-Cit and Arg and ultimately screen for the optimal ratio of L-Cit and Arg.

Cit malate

A major confounding factor in the literature is the interchangeable use of pure L-Cit and Citrulline Malate (CM). From a historical and pharmacological perspective, it is worth noting that when CM was initially developed, malic acid was regarded as the main active ingredient, while L-Cit was merely used as a carrier for obtaining pharmaceutically stable salts. It was not until later that the interest in science and sports nutrition shifted to the physiological effects of L-Cit. CM is a salt form typically provided in a 1:1 or 2:1 ratio. It is crucial to note that Malate is a TCA cycle intermediate that may independently contribute to aerobic energy production and lactate clearance⁹⁰. Therefore, performance benefits observed in studies utilizing CM cannot be solely attributed to Citrulline-mediated NO production. Research indicates that immediate Citrulline malate supplementation can elevate maximal strength and anaerobic capacity in tennis players⁹¹ (Table 1). Moreover, such supplementation has been shown to enhance the performance of female weightlifters engaged in resistance training⁹² (Table 1). However, some evidence suggests that the positive impact of Cit malate on muscles may be limited. Studies have indicated that supplementation with Cit malate does not enhance muscle strength in both healthy individuals and those engaged in resistance training⁹³. Research has shown that acute supplementation with Cit malate does not yield benefits in well-trained healthy males⁹⁴. In healthy individuals participating in recreational training, daily supplementation with 8 g of Cit malate does not significantly reduce muscle fatigue or enhance exercise performance. The efficacy of Cit malate remains uncertain, with current research primarily examining its impact on muscle exercise performance. Its potential effects on muscle mass and protein synthesis are not well-studied. Future research should compare Cit alone with Cit malate to assess whether malate offers additional benefits for exercise performance, muscle mass, and protein synthesis.

L-Cit + Nitrate

Nitric oxide (NO) is a gaseous signaling molecule produced through the Cit-Arg-NO pathway, which depends on the activity of nitric oxide synthase (NOS). Alternatively, NO can be produced through the single-electron reduction of nitrite from dietary sources⁹⁵. Supplementing nitric oxide precursors, such as arginine and nitrate, can enhance the bioavailability of nitric oxide throughout the body⁹⁶. NO regulates vascular tone by activating soluble guanylate cyclase in vascular smooth muscle, essential for muscle contraction and exercise performance. Animal studies indicate that although nitrate might not avert muscle atrophy, it can maintain mitochondrial bioenergetics and temporarily support mitochondrial protein synthesis during short-term muscle immobilization⁹⁷. Further research is needed to clarify the effects of L-Cit, nitrate, or their combination on muscle atrophy and protein synthesis.

However, the applicability of these findings to humans is unclear. In human clinical trials, researchers often supplement with L-Cit, nitrate, or both to evaluate their effects on exercise performance. A 9-week supplementation regimen of 3 g/day L-Cit and 2.1 g/day nitrate-rich beetroot extract failed to reduce muscle damage in triathletes but showed promise in attenuating cortisol levels, potentially aiding recovery⁹⁸. In contrast, a recent meta-analysis suggests that nitrate-rich dietary sources provide minimal benefits for endurance sports, especially for highly trained athletes⁹⁹. Future studies should explore the individual or combined effects of L-Cit and nitrate on muscle performance, taking into account differences in participant profiles, including both well-trained athletes and recreational exercisers. Current literature suggests that nitrate intake does not enhance postprandial muscle protein synthesis in elderly individuals with type 2 diabetes¹⁰⁰.

The interplay between age, metabolic state, and the efficacy of L-Cit

Research on young male amateur runners (mean age 35.3 ± 11.4 years) indicates that consuming 500 ml of L-Cit-rich watermelon juice two hours before a half marathon may improve exercise performance¹⁰¹ (Table 1). Another study with young male participants (mean age 22.2 ± 2.4 years) supplemented with L-Cit and glutathione (2 g L-Cit + 200 mg glutathione) suggests L-Cit can enhance endurance performance¹⁰² (Table 1). Additionally, a single 12 g dose of L-Cit before exercise was found to boost antioxidant markers in young men (mean age 21 ± 0.41 years) after high-intensity interval training¹⁰³.

When evaluating muscle protein synthesis (MPS), age is often considered a primary variable influencing L-Cit’s regulatory effects. In a study of 57 healthy young adults (mean age 24.2 ± 3.3 years), oral L-Cit supplementation improved nitrogen balance by 10%³⁸. Another study with young participants (mean age 25.9 ± 2 years) showed a 25% increase in muscle protein synthesis, especially in those with low protein intake³⁵. Furthermore, a week-long regimen of 6 g/day L-Cit enhanced muscle oxygenation and exercise performance in young males (mean age 19 ± 1 years)¹⁰⁴.

In contrast, L-Cit supplementation (10 g) did not boost myofibrillar protein synthesis in elderly men (aged 65-80 years)¹⁰⁵. This lack of effect in the elderly cohort can be explained by three key protocol divergences compared to successful trials (e.g., Jourdan et al.)³⁵. First of all, the elderly are in good health and well-nourished. Secondly, L-Cit is administered together with the intact protein rather than alone. Because L-Cit is basically effective only under metabolic stress - especially when the Gibbs free energy of adenosine triphosphate decreases - the co-intake of protein may alleviate this energy deficiency, thereby eliminating the anabolic effect of L-Cit. Thirdly, the pharmacokinetic characteristics vary greatly: although successful regimens use continuous administration to maintain persistently high L-Cit levels³⁵, the elderly study utilized a single bolus, causing plasma levels to return to baseline rapidly¹⁰⁵.

Conversely, when elderly subjects are in a stressed metabolic state, L-Cit proves highly effective. Bouillanne et al. demonstrated that in malnourished elderly women (aged 80–92), 10-g/day L-Cit for three consecutive weeks successfully increased actual lean mass and appendicular skeletal muscle mass (ASMM)¹⁰⁶. Interestingly, despite these morphological gains, their research reported no significant increase in systemic protein synthesis. This methodological paradox must be explained through the lens of protein turnover between organs. Just as Osowska et al. established in the malnutrient-induced aging model¹⁵, the unique pharmacokinetic characteristics of L-Cit enable it to largely evade splanchnic extraction. Therefore, L-Cit bypasses the liver to specifically stimulate protein synthesis in skeletal muscle. Because the basal protein turnover in the liver is abnormally high compared to skeletal muscle (about 2–3%), accounting for approximately 40% of the total turnover in the body, the strong and targeted increase in muscle anabolism can easily be masked in whole-body measurements. However, Bouillanne et al. provided indirect evidence for this specific metabolic shift towards muscle: supplementation of L-Cit led to a trend of reduced visceral extraction and a significant increase in the systemic bioavailability of amino acids, ultimately resulting in the observed accumulation of muscle mass¹⁰⁶.

Ultimately, these findings support that metabolic stress rather than youth is a prerequisite for the efficacy of L-Cit. Therefore, L-Cit remains effective in the elderly population, provided that they are in a state of deprivation or stress metabolism. Future research should explore the optimal continuous dosing strategy in the elderly, ensuring that the test is conducted under appropriate metabolic conditions (for example, without co-consuming large amounts of protein), and considering the gender differences resulting from metabolic changes between men and women¹⁰⁷.

The efficacy of L-Cit is influenced by training status and methodological heterogeneity

Consuming watermelon juice rich in L-Cit has been shown to boost metabolism during physical activity, enhance exercise performance, and reduce muscle soreness¹⁰⁸. Research indicates that oral intake of L-Cit and Arg improves athletic performance and decreases muscle discomfort in male soccer players⁸⁸. Similarly, a study with male college track and field athletes found that consuming 3 g/day of L-Cit for seven days improved performance and reduced fatigue¹⁰⁹. Gonzalez et al.¹¹⁰ recently reported a modest improvement in cycling performance among well-trained athletes after L-Cit supplementation.

In elderly overweight individuals without prior training, combining exercise with L-Cit supplementation enhances muscle strength but not muscle mass¹¹¹. Recent studies, however, suggest that untrained males do not experience improved muscle recovery after high-intensity resistance exercise with L-Cit supplementation¹¹². This implies that L-Cit’s benefits may depend on synergy with exercise or resistance training. However, it is important to note that even though L-Cit has potential benefits, providing a clear consensus is still very complex. This difficulty mainly stems from the significant methodological heterogeneity in the current research. Specifically, there are significant differences among the study populations (ranging from outstanding young athletes to overweight elderly people), the different parameters measured (such as metabolism, muscle strength, and perceived fatigue), and the different forms of intake (including pure L-Cit, citrulline malate, L-Cit combined with L-Arg, and watermelon juice). Thus, it is premature to conclude that L-Cit enhances muscle fatigue recovery post-resistance exercise. Further research is needed to investigate its effects on both elite athletes and untrained individuals.

The mechanism by which L-Cit promotes muscle protein synthesis

To clearly understand the mechanism pathways of L-Cit, this section synthesizes current evidence ranging from basic cell models to physiological animal models, and finally to systematic clinical relevance.

Molecular mechanisms in muscle cells

At the cellular level, in vitro studies on catabolic muscle ducts have shown that L-Cit directly regulates muscle protein turnover by simultaneously increasing protein synthesis rate and significantly reducing protein catabolism⁸³. This protective effect seems to be independent of L-arginine conversion; On the contrary, it is mediated by the induction of the inducible nitric oxide synthase (iNOS) isomer, which can prevent muscle cells from wasting¹¹³ (Fig. 3). The combination of in vitro and ex vivo models indicates that L-Cit acts as a direct signaling agent for muscle homeostasis through the mTORC1/PI3K/MAPK pathway. Specifically, L-Cit enhances the phosphorylation of S6K1 and 4E-BP1, and this effect is completely nullified by specific inhibitors such as rapamycin⁶⁷. Cellular energy dynamics also play an important role. Recent in vitro studies using mouse myoblasts have shown that under stress conditions, L-Cit increases muscle protein synthesis by specifically redistributing ATP produced by mitochondria into the protein synthesis mechanism without altering the overall cellular ATP/ADP ratio⁵⁴. Furthermore, proteomic analysis indicates that amino acids such as L-Cit differently regulate the muscle secretome and alter the secretion patterns of specific proteins¹¹⁴. Future in vitro studies should explore this transformation of the secretome to discover new local functions.

Fig. 3. Possible mechanism of citrulline promoting MPS.

MPS muscle protein synthesis, Cit citrulline, PGC-1α peroxisome proliferator-activated receptor γ coactivator 1α, mTOR mechanistic target of rapamycin, PI3K phosphatidylinositol 3-kinase, AKT Akt kinase, S6K1 S6 kinase 1, 4EBP1 4E-binding protein 1, TFAM mitochondrial transcription factor A, MEK1/2 MAP kinase kinase 1/2, ERK1/2 extracellular signal-regulated kinase 1/2. Illustration created with BioRender.com.

Molecular mechanisms in animal models

In vivo animal studies have demonstrated how L-Cit regulates the systemic network to support muscle tissue. In elderly or malnourished rat models, L-Cit supplementation effectively activates muscle protein synthesis, and muscle S6K1 phosphorylation shows a positive correlation with plasma L-Cit concentration⁶⁷. This systemic administration beneficially alters body composition by increasing lean muscle mass and preventing lipid oxidation¹⁹. Although muscle proteins include various types¹¹⁵, in vivo evidence emphasizes that endurance training combined with L-Cit can synergically increase muscle protein synthesis and regulate energy metabolism through the acetyl-CoA pathway⁶¹. In addition, the animal model reveals the L-Cit role in angiogenesis and mitochondria biological generation. In mice, oral L-Cit supplements significantly upregulated the expression of peroxisome proliferator-activated receptor -γ coactivator -1α (PGC-1α), and subsequently upregulated VEGFα and IGF-1. This angiogenic effect improves blood flow and prolongs exercise time until exhaustion⁵⁶. In addition, L-Cit is associated with an increased expression of mitochondrial transcription factor A (TFAM) in vivo¹⁹ (Fig. 3).

Systemic regulation and clinical relevance: the Gut-Muscle axis

In vivo clinical trials on healthy volunteers and patients with short bowel syndrome demonstrate that while oral L-Cit supplementation significantly increases plasma L-Cit and ARG bioavailability, it does not alter whole-body protein kinetics^116,117. L-Cit’s anabolic effects are highly localized to specific tissues like skeletal muscle, deeply connected to the L-Cit-gut microbiota-muscle axis¹¹⁸.

Clinically, systemic plasma L-Cit serves as a reliable biomarker for functional enterocyte mass. For instance, in leukemia patients undergoing chemotherapy, severe gut damage correlates with a sharp drop in plasma L-Cit and pronounced dysbiosis¹¹⁹. Moreover, human population studies show significant variations in gut microbiota (e.g., Prevotella abundance) among Indian, American, and Jamaican women, which strongly correlate with differing rates of L-Cit and arginine metabolism¹²⁰.

Appropriate supplementation of watermelon products rich in L-Cit in mice can change the composition of the microbiome and improve metabolic parameters¹²¹. In vitro intestinal models also demonstrated that L-Cit and probiotics (such as Lactobacillus Swiss) have a synergistic effect in enhancing the intestinal epithelial barrier against pathogens¹²². Interestingly, while probiotics like Lactococcus lactis can stimulate endogenous L-Cit production in aquatic models¹²³, rumen microorganisms in adult steers do not degrade L-Cit, proving its unique microbial stability¹²⁴. Since gut health dictates L-Cit bioavailability, manipulating the gut microbiota to optimize the L-Cit-gut-muscle axis represents a crucial clinical strategy for counteracting muscle wasting.

Development of L-Cit complexes: future trends and market prospects

Limitations of L-Cit use

The application of L-Cit is currently limited by specific formulations and conveying challenges. First of all, the stability of L-Cit highly depends on its physical state. Although pure L-Cit in powder form is extremely stable, L-Cit naturally present in aqueous plant substrates, such as the pulp of cucumbers and watermelons, can significantly degrade during cold storage, thereby reducing the effective content in the final product¹²⁵. Furthermore, although L-Cit typically exhibits a high absorption rate by bypassing the liver’s first-pass metabolism, its free form still faces local instability in the harsh environment of the gastrointestinal tract, which may lead to suboptimal controlled release¹²⁶. Consuming large amounts of L-Cit from natural sources such as watermelon may impose a burden on the digestive system, especially for people with impaired kidney function¹²⁷.

Design strategies for L-Cit complexes

By taking advantage of the metabolic characteristics of L-Cit and the existing research on amino acid chelates, we can develop L-Cit complexes to improve energy metabolism. These complexes require careful consideration of factors such as ligand selection, chelation mode, stability, and bioavailability.

L-Cit-trace element chelates

Amino acid chelates are stable compounds characterized by a cyclic structure formed through coordination bonds between amino acids and metal ions¹²⁸. Although naturally occurring, they are industrially synthesized via precise chemical or biocatalytic methods. Their stability and bioavailability arise from the metal ions’ coordination with amino and carboxyl groups, creating a stable five- or six-membered ring¹²⁹. This structure ensures the metal elements remain soluble in physiological pH environments until they are delivered to specific sites. The global market for amino acid chelates is rapidly expanding, with significant growth in agriculture, feed additives, and human nutrition and health, where their applications are continually evolving.

However, as a chelating agent, L-Cit has chemical limitations compared with traditional amino acids such as glycine or methionine. Specifically, the urea group on the side chain of L-Cit remains uncharged at physiological pH and shows a low affinity for metal ions in aqueous solutions. In addition, the significant steric hindrance imposed by the propyl urea side chain will make the chelating ring unstable. Therefore, L-Cit mainly acts as a simple bidentate ligand, resulting in a relatively weak metal coordination ability and a lower stability constant¹³⁰. To overcome these limitations, L-Cit may be developed through the development of mixed ligand (ternary) complexes. The coordination sphere can be thermodynamically stable by introducing secondary small molecule ligands (such as malate or glycine). This strategy may be able to counteract the weak chelating ability of L-Cit, ensuring the solubility and targeted delivery of trace elements while maintaining its synergistic metabolic benefits^131,132.

Trace elements are crucial cofactors in energy metabolism¹³³. When chelated with L-Cit, they may exhibit synergistic effects (Fig. 4). Zinc is a prime candidate, participating in the mTOR pathway and influencing over 300 enzyme activities^134,135. Zinc deficiency can impair ATP synthesis¹³⁶. The L-Cit-zinc chelate might synergistically enhance anabolism and could be particularly effective against sarcopenia, though this remains unverified. Magnesium is also noteworthy, serving as an essential cofactor for ATPase and directly catalyzing ATP hydrolysis to release energy¹³⁷. The L-Cit-magnesium chelate could potentially elevate intracellular magnesium levels, boosting energy turnover in high-demand tissues like the myocardium and skeletal muscles. Additionally, the L-Cit-chromium chelate holds promise. Chromium enhances insulin sensitivity¹³⁸ and promotes glucose uptake and, combined with L-Cit’s influence on glucose metabolism, may be an apt supplement for individuals with metabolic syndrome. However, the specific formula of L-Cit-trace element chelates is still entirely theoretical. It must be pointed out that, to the best of our knowledge, there is currently a lack of direct preclinical or clinical data to prove the biological benefits of chelating L-Cit with mineral salts.

Fig. 4. Future trends and market prospects of L-Cit complexes.

The use of free L-Cit is constrained by challenges related to stability, bioavailability, dose control, and digestive issues. Advances in L-Cit complexes, such as L-Cit-Trace Element Chelates with cyclic metal ion structures and formulations with flavonoids and polysaccharides, seek to improve stability, bioavailability, and energy metabolism. Illustration created with BioRender.com.

L-Cit complex

As with trace element chelates, it must be clearly pointed out that the specific combinations of L-Cit with phytochemicals or polysaccharides discussed below are currently valid hypotheses. There is a lack of direct preclinical or clinical data to prove the synergistic biological benefits of these specific L-Cit complexes.

Flavonoids, prevalent in plants and known for their diverse biological activities, are linked to energy metabolism¹³⁹. When combined with L-Cit, they may exhibit synergistic effects. Citrus flavonoids, such as hesperidin and naringin, are effective as ligands (Fig. 4). These compounds inhibit phosphodiesterase, elevate cAMP levels, and enhance fat oxidation^140–142. Additionally, they suppress inflammatory mediators like TNF-α^143,144, thereby disruptions to energy metabolism. The vasodilatory and anti-inflammatory properties of these flavonoids complement those of L-Cit, potentially enhancing exercise endurance and recovery^145,146. Epicatechin, found in green tea, is another flavonoid that activates the AMPK pathway and induces mitochondrial biogenesis^147,148. This interaction with L-Cit may synergistically engage AMPK-regulated energy metabolism, allowing the complex to target the energy-sensing pathway.

Polysaccharides are natural macromolecular compounds with good biocompatibility and various physiological activities. The complex formed by β -glucan (derived from oats or mushrooms) and L-Cit has obvious advantages. β -glucan can regulate macrophage metabolism¹⁴⁹, inhibit inflammatory responses¹⁵⁰, and may enhance L-Cit absorption by improving intestinal barrier function¹⁵¹. This complex is expected to address low energy metabolism problems caused by chronic inflammation, such as age-related fatigue, and may prolong the efficacy of L-Cit. The formation of complexes may involve embedding or molecular binding, rather than just chelation. For instance, coating L-Cit with chitosan and nano-liposomes can not only enhance its stability but also regulate its release in the gastrointestinal tract, thereby increasing its bioavailability¹²⁶.

Designing these complexes involves addressing technical challenges such as ensuring stability, optimizing the preparation process, and managing costs. Furthermore, absorption, metabolism, and bioavailability must be validated through rigorous experimentation. Despite the challenges, the design of these complexes offers broad prospects for L-Cit applications, especially in precision nutrition and health. The successful application of amino acid chelates in various fields provides a reference for the development of L-Cit chelates. In the feed industry, amino acid chelated minerals are recognized, including methionine, glycine, and threonine chelates, and are widely used in ruminants, pigs, poultry, and aquaculture^152–156. They offer dual nutritional benefits by providing amino acids and trace minerals. In terms of human nutrition, the use of amino acid chelates is expanding, being applied in dietary supplements, formula milk powder, special medical foods, and sports nutrition products. These products take advantage of the high bioavailability and low gastrointestinal irritation of amino acid chelates to meet specific nutritional needs.

Conclusions and perspectives

L-Cit is a potential supplement for enhancing muscle function and protein synthesis. Its effectiveness depends on factors such as the supplementation method, age, and exercise plan. Future studies should identify potential L-Cit transporters in intestinal epithelial and skeletal muscle cells. The computational model using plasma L-Cit concentration and mRNA expression data can simulate the transient L-Cit levels in these cells. Furthermore, it is of vital importance to study the combined effects of L-Cit and other supplements on muscle function and protein synthesis, as well as the regulatory effect of L-Cit on mitochondrial function. To enhance the stability of L-Cit in water environments and its suboptimal controlled release in the gastrointestinal tract, the L-Cit complex hypothesis is proposed by creating mixed ligand (ternary) complexes or combinations with flavonoids and polysaccharides. However, these combinations currently lack direct preclinical or clinical validation and require rigorous experimental evaluation. Finally, the L-Cit-gut-microbiota-muscle axis deserves attention (Fig. 5).

Fig. 5. L-citrulline (L-Cit) holds promise as a supplement for boosting muscle function and protein synthesis, though its efficacy is modulated by factors like supplementation methods, age, and exercise routines.

Future studies should aim to identify L-Cit transporters in intestinal epithelial and skeletal muscle cells and model cellular L-Cit levels using plasma concentration and mRNA data. The synergistic effects of L-Cit with other supplements and its role in mitochondrial function regulation warrant further exploration. Novel L-Cit complexes, such as those with flavonoids and polysaccharides, might enhance stability and bioavailability. Additionally, the citrulline-gut-microbiota-muscle axis is a critical area for ongoing research. Illustration created with BioRender.com.

Author contributions

Xiaokang Lv: Wrote the paper. Jinling Hua: Revised the manuscript. Xiaokang Lv: conceptual design, project management. Data collection: Yating Song and Tian Guo. All authors read and approved the final manuscript.

Data availability

No datasets were generated or analysed during the current study.

Competing interests

The authors declare no competing interests.