1. Introduction
Prolonged and strenuous exercise produces organic stress [
1] that could decrease athletic performance [
2,
3,
4]. As a consequence of this status, there are several alterations in biochemical parameters of exercise-induced muscle damage (EIMD) [
5] as well as anabolic/catabolic hormone alterations which could hinder endogenous exercise adaptations [
6]. Therefore, in addition to an adequate training program, it could be essential to include different strategies to delay or reduce muscle fatigue and improve adaptation to training [
7]. In this sense, supplementation with nitrate-rich beetroot extract (BR) and citrulline (CIT) has been proposed to achieve these goals, partly because they are precursors of nitric oxide (NO) [
8,
9,
10,
11].
The NO produces vasodilation by increasing the blood level in muscles and improving their efficiency in muscle contraction and relaxation processes [
12]. Moreover, NO regulates force generation and satellite cell activation [
13]. In the long term, NO can regulate muscle function and even affect skeletal muscle recovery due to its antioxidant effect and the constant increase in muscle blood flow which, together with an adequate supply of essential amino acids, would allow better muscle fueling [
14] and could prevent EIMD [
15,
16]. Moreover, decreased blood flow to the testis could reduce testosterone synthesis [
17]. It has also been shown in animal models that NO enhancement resulted in a significant reduction of ACTH-mediated cortisol production [
18]. Consequently, although this mechanism is speculative, increasing NO could improve blood flow in the testis and promote testosterone synthesis by vasodilator effect [
14,
19] and could be successful in maintaining an anabolic state, decreasing muscular damage and metabolic stress [
2,
9].
On the one hand, BR supplementation is widely used by athletes as a precursor of NO [
20]. When the athletes digest BR, its nitrates (NO
3−) are transformed into nitrites (NO
2−) which are partially reduced to NO by the action of stomach acids and subsequently absorbed in the intestine and passed into the bloodstream [
21]. Moreover, BR is rich in other compounds such as phenolic acids, flavonoids, carotenoids and betalains, which have antioxidant effects [
22]. Therefore, although the mechanisms for potential improvements in muscle recovery following EIMD after NO
3− supplementation are not clear, it would be expected that long-term BR supplementation could attenuate EIMD after prolonged, strenuous exercise [
22,
23] based on the effects of NO and additional compounds. Moreover, long-term BR supplementation could be very beneficial for the maintenance of anabolic/catabolic hormones, as shown by Sarfaraz et al. on testosterone levels [
24]. However, short-term BR supplementation (maximum for 3 days) has not presented an improved EIMD and anabolic/catabolic status after a damaging session of eccentric exercise [
23] or high-intensity workouts [
25], which opens the need for further research.
On the other hand, citrulline (CIT), a non-essential amino acid found primarily in watermelon and produced endogenously by recycling into arginine (ARG) and NO via argininosuccinate synthetase, increases NO availability and its effects [
26]. In addition, CIT is an essential element of the urea cycle in the liver [
27]. Therefore, it has been suggested that CIT supplementation may eliminate ammonia by urea production [
28]. In the same way, CIT is an important activator of muscle protein synthesis in catabolic situations via activation of the mammalian target of rapamycin (mTOR) pathway due to its key role in the regulation of nitrogen homeostasis [
29]. Based on these mechanisms, CIT supplementation may favor muscle performance and recovery in different ways, such as activating muscle protein synthesis, improving oxygen distribution to muscle, increasing oxidative ATP production during exercise and phosphocreatine (PCr) during exercise recovery and decreasing blood lactate and ammonium production [
14,
30,
31], which could reduce fatigue and limit EIMD. However, although this proposal would be adequate for athletes, to the best of the authors’ knowledge, there is little research on CIT supplementation in muscle recovery. In this regard, Da Silva et al. [
27] did not observe improvements in functional (i.e., number of maximum repetitions, muscle pain and perceived effort), metabolic (i.e., CK and lactate), anabolic (i.e., testosterone and testosterone/cortisol (T/C) ratio) and physiological (electromyographic signal) outcomes of muscle recovery in untrained young adult males after CIT supplementation with 6 g at 60 min prior to the training session. These results of both CIT and BR supplementation on EIMD and anabolic/catabolic hormones may probably be due to the fact that the effects have only been investigated in the short term [
28] and under isolated intakes [
29,
32], suggesting the need to investigate the effects of long-term combination of these two ergogenic aids. In this regard, it has been shown that the effects of some supplements can be synergic when combined over the long term [
2,
33]. Therefore, it could be considered that the combined effects of CIT (NO precursor and activator of muscle protein synthesis) and BR (NO precursor and antioxidant effect) could reduce EIMD and improve muscle recovery observed by anabolic/catabolic hormone profile [
34,
35]. This could favor some sporting performance variables [
36]. In this sense, the supplementation of 6 g of CIT plus 520 mg of NO
3− 6 h before the submaximal incremental cycling test has shown improvements in some cardiorespiratory variables, such as VO
2 [
36].
Therefore, the main objective of this research was to assess the effect of the long-term (9 weeks) mixture of 3 g/day of CIT plus 2.1 g/day of BR (300 mg/day of NO3−) supplementation on recovery status, distance covered in the Cooper test, EIMD markers (urea, creatinine, AST, ALT, GGT, LDH and CK) and anabolic/catabolic hormones (testosterone, cortisol and T/C) in male trained triathletes. The hypothesis was that the combination of CIT plus BR could limit EIMD and improve endogenous recovery observed in lower cortisol and better testosterone and T/C than isolated CIT or BR supplementation.
3. Results
During the trial, the triathletes did not present significant statistical differences (
p > 0.05) in energy and macronutrient intake values among groups (
Table 3).
Body mass, BMI, muscle mass and fat mass percentage did not present significant differences (
p > 0.05) in the interaction group-by-time (
Table 4).
Figure 1 shows the distance covered in the Cooper test at both T1 and T2. Significant differences can be seen in the group-by-time interaction in this parameter (
p = 0.002; ƞ
2p = 0.418). In addition, significant increases (
p < 0.05) were observed between study moments in distance covered (T1: 2953.1 ± 372.7 vs. T2: 3079.6 ± 423.5 m) in CIT-BRG.
The EIMD markers did not present significant differences (
p > 0.05) in the group-by-time interaction (
Table 5). However, significant differences were observed between T1 and T2 for BR in creatinine (T1: 0.92 ± 0.11 vs. T2: 0.88 ± 0.09 mg/dL; ƞ
2p: 0.063) and LDH (T1: 445.38 ± 247.59 vs. T2: 393.88 ± 63.37 UI/L; ƞ
2p: 0.083).
Table 5 displays significant differences in the group-by-time interaction for cortisol (
p = 0.044; ƞ
2p = 0.247) and T/C (
p = 0.005; ƞ
2p = 0.359). In this sense, a significant difference was observed for T/C in CIT-BRG with respect to PLG at T2. On the other hand, a significant decrease in testosterone levels and T/C was observed in PLG, CITG and BRG after 9 weeks of supplementation (
Table 6).
Figure 2 shows the percentage change in distance covered in the Cooper test for each of the study groups. Significant differences can be observed in this parameter (
p = 0.002; ƞ
2p = 0.424). Concretely, CIT-BRG presented a significantly higher value in the % change than PLG and CITG (
p < 0.05).
Figure 3 indicates significant differences in cortisol percentage change (
p = 0.049; η
2p = 0.257) between PLG and CIT-BRG. Moreover, T/C percentage change presented statistically significant differences (
p = 0.018; η
2p = 0.297) between CIT-BRG and PLG. In the case of testosterone, there were no significant differences among groups in percentage change (
p = 0.149).
4. Discussion
This study was planned to assess the effect of long-term (9 weeks) combination of 3 g/day of CIT plus 2.1 g/day of BR (300 mg/day of NO3−) supplementation on recovery status by distance covered in the Cooper test, serum EIMD markers and testosterone and cortisol in male triathletes. The EIMD markers (urea, creatinine, AST, ALT, GGT, LDH, CK) did not show any significant differences in the group-by-time interaction. However, triathletes showed a significantly better group-by-time interaction in distance covered in the Cooper test and anabolic/catabolic hormone status in CIT-BRG by preventing an increase in cortisol and a better T/C ratio. Furthermore, while CITG and BRG showed a significant decrease in testosterone levels, CIT + BR supplementation prevented a decline of this anabolic hormone. These significant results could be motivated by the synergistic effect that both supplements provided on the variables used to determine recovery status.
The balance between training loads and recovery are key factors in improving athletic performance [
4]. To assess and control this balance, and with the intention of avoiding fatigue and maintaining an adequate performance, there are numerous parameters utilized, such as EIMD markers and anabolic/catabolic hormones [
57,
58]. Although there is an acute intensification of EIMD markers after exercise [
2,
59], long-term maintenance of high EIMD values could indicate a chronic fatigue status and inadequate adaptation to training [
60]. In addition, it has been observed that anabolic/catabolic hormone status is changed after exercise due to an acute effect [
58,
61]. However, long-term variations in these hormones may be indicators of an adequate endogenous adaptation or, on the contrary, a fatigue status and, therefore, of an impaired sports performance [
6]. Testosterone is an anabolic and androgenic hormone secreted by the hypothalamic–pituitary–testicular axis, and its increase specifies an overall anabolic state [
62]. Nevertheless, cortisol, secreted by the hypothalamic–pituitary–adrenal axis, is a steroid hormone considered as a factor that indicates accumulated stress, and therefore, its increase suggests an accumulation of stress or catabolism [
63]. Consequently, an increase in testosterone and/or a decrease in cortisol would lead to an increase in the testosterone/cortisol ratio, as an indicator of adaptation to training, thus indicating better endogenous recovery, while a decrease would indicate fatigue status [
61,
64]. In order to achieve these effects, some supplements that promote the NO pathway, such as CIT and BR, have been proposed [
31].
It has been shown that NO can enhance recovery status through certain mechanisms [
65], such as increasing protein synthesis through vasodilation of the arteries and veins of skeletal muscle that improve nutrient flow to the muscles, which in the long term favors muscle growth and repair [
66]. In addition, it has been suggested that NO probably promotes angiogenesis in tissues by regulating the expression of the vascular endothelial growth factor [
67]. Moreover, it has been demonstrated that skeletal muscle has the capacity to store, transport and metabolize NO
3− and NO
2− [
68]. Therefore, chronic supplementation with NO precursor supplements (CIT and BR) would increase the levels of NO
3− stored in skeletal muscle that is beneficial for NO production [
69]. All these mentioned mechanisms could probably work in a complementary manner by enhancing endogenous recovery. A more efficient production of energy during exercise would reduce fatigue and thus decrease EIMD through an increase in protein synthesis [
70]. This improved regeneration would lead to a decrease in stress and thus a reduced catabolic state, which would be reflected in anabolic/catabolic hormones [
11].
In addition to the effect on NO, the CIT has been found to stimulate muscle protein synthesis by activating mTOR through the PI3K/MAPK/4E-BP1 pathway [
71] and by increasing ARG production, which promotes growth hormone secretion [
72]. Likewise, increased ARG production will improve intramuscular creatine levels, which will also allow an increase in phosphocreatine reserves, contributing to energy supply through a more efficient ATP regeneration and lowering fatigue, resulting in a decrease in EIMD after a high-demanding training [
31]. Moreover, being part of the urea cycle, CIT facilitates the functioning of this cycle, helping to reduce the accumulation of ammonium and blood lactate concentration, improving the clearance capacity of these substances and, therefore, reducing the fatigue caused by their accumulation [
73].
To the authors’ knowledge, the effects of long-term combination of CIT plus BR supplementation on EIMD markers have not been studied in depth [
74]. In this sense, the present trial did not present significant differences in the interaction between group and time in EIMD markers (urea, creatinine, AST, ALT, GGT, LDH and CK) (
p > 0.05;
Table 4). In the same line, some investigations that have evaluated the acute effects of these supplements individually have not found improvements in EIMD markers. Daab et al. did not find significant differences in CK and LDH before the Loughborough Intermittent Shuttle Test between the supplemented and the placebo groups after 7 days (3 days pre-exercise, test day and 3 days post-exercise) with 150 mL of BR juice (250 mg of NO
3−) taken in two intakes per day (08:00 and 18:00 h) in soccer players [
74]. Likewise, Martínez-Sanchez et al. did not present significant differences in the biochemical markers AST, ALT and CK between the supplemented group and placebo with CIT-enriched watermelon juice (3.45 g per 500 mL/day) taken two hours before a half-marathon race [
66]. Therefore, considering that the results of this study did not offer any beneficial effects on the EIMD markers, the results obtained by the combination of CIT plus BR in the present study dismantle the original hypothesis in which it was predicted that both supplements could work in a complementary manner by reducing EIMD.
Although, to the authors’ knowledge, the effects of long-term combination of CIT plus BR supplementation on anabolic/catabolic hormones have not been studied, in the current study, the combination of these supplements showed a better group-by-time interaction in distance covered in the Cooper test and anabolic/catabolic hormone status in CIT-BRG (
Table 5 and
Figure 3) by preventing an increase in cortisol (
p = 0.044; η
2p = 0.247) and a better T/C ratio (
p = 0.005; η
2p = 0.359). Furthermore, while CITG and BRG showed a significant decrease in the testosterone level after 9 weeks (
p < 0.05), CIT + BR supplementation prevented a decline in this anabolic hormone. Nevertheless, some authors have shown the effect of both supplements individually on these hormones. In this way, Da Silva et al. did not observe improvements in the T/C ratio during the recovery period at 24, 48 and 72 h post-exercise in untrained young adult men after 6 g CIT supplementation before a 60 min workout [
27]. These authors indicated that the inability to improve anabolic factors results in no beneficial effect of CIT supplementation on muscle regeneration during an acute recovery period. However, the chronic changes in cortisol and testosterone can be related to accumulated stress and body regeneration during the sports season [
6]. In this way, Garnacho-Castaño et al. showed that BR supplementation did not appear to influence anabolic/catabolic status in response to acute high-intensity workouts after drinking 140 mL of BJ (~12.8 mmol NO
3−) [
25]. On the contrary, in this study, CITG and BRG presented a maintenance of distance covered in the Cooper test and a decrease in testosterone levels and T/C after 9 weeks of supplementation. These data could indicate an inadequate recovery status in these groups because after 9 weeks with adequate training, a better sports performance would be expected. Nevertheless, this study presented a significantly better recovery status in CIT-BRG represented as an increase in distance covered in the Cooper test and maintenance of testosterone and T/C ratio after 9 weeks of combined supplementation. These adaptations were obtained by preventing an increase in cortisol and/or a decline in testosterone in CIT-BRG with respect to other supplementation groups.
In this sense, CIT is a key activator of muscle protein synthesis in catabolic situations, such as high-intensity training periods, via activation of the mTOR pathway due to its key role in the regulation of nitrogen homeostasis [
75]. Likewise, testosterone increases mTOR pathway [
76] and cortisol inhibits mTOR pathway signaling [
75]. Thus, increasing testosterone and controlling cortisol secretion could result in lower stress and adequate muscle regeneration [
6]. In this sense, the long-term effect of CIT enhancing NO could increase blood flow in the testis promoting testosterone synthesis [
19] and maintaining the testosterone level by vasodilator effect [
77]. In addition, the enhancement of NO reduces ACTH-mediated cortisol production [
18]. Consequently, although this hypothesis is speculative, increasing NO could be successful in maintaining an anabolic state, decreasing metabolic stress [
2,
9]. Therefore, the results obtained in the CIT-BRG group could show how independent pathways in muscle recovery (NO and mTOR) can be synergistically activated with both supplements to obtain better results.
4.1. Limitations, Strengths and Future Research
It should be noted that it is difficult to obtain larger samples in athletes as not many of them have the availability to comply with the training and supplementation instructions required by the study. In addition, the effects that both supplements used could have on the muscle were speculative because no evaluation was included in this regard. On the other hand, sampling using a convenient, non-probabilistic sampling procedure may produce results that are not representative of the rest of the population. These limitations may underrepresent the results and may affect study outcomes. For this reason, the results should be considered in the context of the study. However, the methodology used in this trial, a double-blind, placebo-controlled trial, is the most important strength. In addition, another strength was the control of the triathletes’ diet, as well as the control of the body composition throughout the intervention process, so that these outcomes did not influence the final results. Another strength is the synergistic potential of the study.
Future research should continue to study the long-term effects of this combination on recovery, using different markers, such as sports performance, in order to expand the existing knowledge on this combination. It should also examine the effectiveness of these supplements in athletes who have already been diagnosed with an overtraining state to determine whether the use of these supplements as part of treatment would accelerate recovery. In addition, it should analyze how this potential combination affects the female population or anaerobic sports, given that this study only focused on males and measured aerobic performance.
4.2. Practical Application
This research could be of interest to physicians and nutritionists who want to achieve better post-exercise recovery for their athletes. Considering that 3 g/day of CIT plus 2.1 g/day of BR (300 mg/day of NO3−) for 9 weeks could advance muscle and endogenous recovery, supplementation phases could be considered in the intensive training phases.