Impact of a short-term nitrate and citrulline co-supplementation on sport performance in elite rowers: a randomized, double-blind, placebo-controlled crossover trial

Aerobic performance and maximal power have been proposed as the 2 main physiological determinants and predictors of rowing performance (Ingham et al. 2002; Izquierdo-Gabarren et al. 2010). Aerobic performance can be explained by central and peripheral factors determining the capacity to sustain power production for a given time (Coyle 2007). Among them, whole-body peak oxygen uptake (VO_2peak) is a widely used outcome associated with performance and exercise capacity and determines the aerobic capacity of an individual (Bassett and Howley 2000). In professional rowers, the VO_2peak is positively correlated with performance during high-intensity endurance efforts lasting between 5 and 7 min (Papadakis et al. 2022; Penichet-Tomas et al. 2023). VO_2peak is partly determined by the capacity of the heart to provide blood to peripheral tissues—explained by the stroke volume and maximal heart rate—and therefore, maximum heart rate has been always considered a critical outcome (Coyle 2007). Moreover, aerobic performance can be also explained by the ability to oxidize substrates at cellular level—the so-called mitochondrial function—that is commonly assessed in sports field by the blood lactate concentration due to its inverse correlation with fatty acid oxidation and mitochondrial respiration (San-Millán and Brooks 2018; Brooks 2020). In addition, the ability to produce and maintain peak stroke power is positively associated with muscle strength (maximal strength) (Bell et al. 1989) and related to performance (the higher is the power production—high peak power—and maintenance—high mean power and low fatigue index—, the higher the rowing performance) (Lawton et al. 2013).

Both main physiological determinants are usually improved with training and nutritional interventions and optimized to a greater level with additional interventions (i.e., use of supplementation products) that athletes rely on (Jeukendrup 2017; Tiller et al. 2019). Nitric oxide synthase-related supplements such as nitrate-rich beetroot (BR) extract, or amino acids such as citrulline (CIT) and arginine, have garnered attention due to their potential beneficial influence on outcomes related to performance (Bescos et al. 2012; Bryan 2018; Maughan et al. 2018). Nitrate serves as the precursor of nitric oxide (NO), an important bioactive molecule that is produced endogenously from L-arginine, as extensively described elsewhere (Lundberg et al. 2008; Milton-Laskibar et al. 2021). Briefly, NO production from precursors’ supplementation produced positive effects that could positively influence sports performance (Bailey et al. 2009; Bescos et al. 2012; Jones 2014a; Dominguez et al. 2018; Gonzalez and Trexler 2020; Viribay et al. 2020).

Supplementation with BR elevates circulating nitrate (NO₃⁻) levels and the production of NO (Milton-Laskibar et al. 2021). BR’s most remarkable effects are vascular vasodilation, increasing blood flow supply and tissue oxygenation (Clements et al. 2014), and acute or chronic BR supplementation has shown positive effects on performance. Previous literature showed that BR ingested 2.5 h before exercise improved aerobic power by reducing the total time to complete a 4 km time trial in cyclists (Lausch et al. 2017). Similar effects were observed in highly trained rowers ingesting this supplement 2 h before exercise (Hoon et al. 2014). Concerning short-term ingestion (8 days of daily BR intake), a previous study showed no improvement on a 1500 m time trial in elite runners (Boorsma et al. 2014). Conversely, in a sample of trained rowers, 6 days of daily BR intake improved exercise performance after consecutive 6 × 500 m trials (Bond et al. 2012). In addition, BR seems to improve human muscle contractile capacity (Coggan and Peterson 2018; Coggan et al. 2021) and could positively influence muscle strength and peak force production. A previous study performed in trained adolescents showed improvements in all-out Wingate test-related parameters such as peak power, average power, and fatigue index after BR intake 2.5 h before exercising (Bender et al. 2018). Daily supplementation of BR for 6 days improved resistance-trained males’ strength and resistance exercise performance (Mosher et al. 2016). Nevertheless, differences between studies are large and that could be related to the amount of BR intake (BR amount ranged from 5.5 to 19.7 mmol) which arouses the necessity to study the potential correlation between effects and supplementation dosage (Bond et al. 2012; Hoon et al. 2014; Boorsma et al. 2014; Mosher et al. 2016; Lausch et al. 2017; Coggan and Peterson 2018; Bender et al. 2018; Coggan et al. 2021). The CIT is an amino acid that possesses potential ergogenic properties, as extensively described elsewhere (Curis et al. 2005; Wu et al. 2009). CIT supplementation is supposed to reduce the accumulation of blood lactate and acidosis during high-intensity exercise, which may result in enhanced aerobic performance and muscle strength production (Rhim et al. 2020). In trained males, CIT consumed 1 h before exercise reducing the time necessary to cover 4 km (Suzuki et al. 2016). Similarly, in healthy males and females, CIT consumed 3 h before exercise increasing the time until exhaustion (Hickner et al. 2006). Concerning muscle strength production, CIT supplementation could stimulate muscle protein synthesis (Goron et al. 2019). For instance, acute ingestion of CIT 1 h pre-exercise favored improvements in the strength production capacity in both trained and untrained males (Pérez-Guisado and Jakeman 2010; Wax et al. 2015; Trexler et al. 2019). Despite the potential effects of aforementioned studies, two recent systematic review and meta-analysis studies suggested that CIT supplementation might not influence aerobic exercise performance (Viribay et al. 2022) and muscle strength production capacity (Aguiar and Casonatto 2022). However, a wide range of intensities (60–100% VO_2max) were included in one meta-analysis (Viribay et al. 2022) and only 4 studies were included in the remaining one (Aguiar and Casonatto 2022), suggesting data around effects of CIT on VO_2peak and strength is still scarce. Nevertheless, as BR and CIT supplements elevate NO production (Bescos et al. 2012), the study of their ergogenic effects on athletes’ performance deserves attention (Lansley et al. 2011; Hernández et al. 2012; Haider and Folland 2014). Nowadays, an emerging field of study is the combination of BR and CIT supplements. In fact, it is plausible to hint at potential synergic benefits with the combination of BR and CIT (Roux-Mallouf et al. 2019), and other NO-related supplements such as CIT and arginine (Suzuki et al. 2019). In this regard, improvements in aerobic power performance and maximal and endurance strength have been found after 9 weeks of combined supplementation in trained miles (Burgos et al. 2021). Moreover, same supplementation protocol confirmed enhanced recovery status (biochemical parameters) and performance in aerobic power test (Burgos et al. 2022). However, available literature on the combined supplementation of BR and CIT using different intake durations (e.g., long-time intake supplementation vs. acute intake supplementation), and across sport modalities is still limited. Thus, their potential effect on performance deserves attention.

The main objective of this study was to determine the effects of short-term intervention (1 week) with oral co-supplementation of BR (3.5 g per day) extract (500 mg NO₃⁻ per day) plus CIT (6 g per day) on aerobic performance (power output, VO_{2 peak}, lactate clearance and heart rate), by graded incremental test, maximal strength by counter-movement jump test (height), and high-intensity power and peak stroke by Wingate test (mean, relative and peak power, and fatigue index) in elite male rowers compared to a placebo and to a BR supplementation (1 week each).

A total of 20 men (29 ± 7 years old, 13 ± 6 rowing experience) participated in the study. Additional participants’ characteristics are presented in Table S1. Of note, body weight remained unaltered (P > 0.05) across study conditions (Table S1).

Finally, no between-conditions differences were observed in blood samples (all P > 0.200), as all blood outcomes remained unaltered between study conditions (Table S4).

This study aimed to assess the impact of short-term chronic (1 week) supplementation with oral co-ingestion of 6 g of CIT and 3.5 g of BR extract (500 mg or 8 mmol NO₃⁻) per day on maximal strength (CMJ jump test), high-intensity power (mean, relative and peak power, and fatigue index in Wingate test), and aerobic performance (by analyzing power output, VO_{2 peak}, lactate clearance and heart rate) in elite male rowers, compared with BR extract taken alone and PLA. The results showed no between-conditions (PLAG, BRG, and BR-CITG supplementation intervention) differences in Wingate test-related outcomes. However, 5-min post-Wingate test blood lactate concentration levels showed a significant difference in BR-CITG condition compared to BRG and PLAG conditions, thus suggesting a better post-exercise lactate clearance. Furthermore, differences were found in peak power during the aerobic performance test in BR-CITG compared to PLAG. Nonetheless, other related physiological parameters such as VO_2peak and maximum heart rate showed no differences. No other differences were observed in CMJ test-related outcomes and fasting blood circulating markers. Our results might confirm the proposed synergic effects of BR-CITG supplementation.

NO is an important bioactive molecule that has been related to improved oxygen (O₂) delivery by impacting outcomes such as VO₂ max, exercise economy, and fractional utilization of the VO₂ max (Jones 2014b). As NO₃⁻ and CIT serve as precursors of endogenous NO production (Lundberg et al. 2008; Milton-Laskibar et al. 2021), supplements containing NO₃⁻ such as BR extract and CIT abundant supplements like citrulline-malate have been studied to understand potential effects on O₂ delivery. Larsen et al. (2007) studied for the first time the impact of 3 days of sodium nitrate supplementation (0.1 mmol/kg body weight) in VO_{2 max} and no effects were found, but surprisingly a reduction of 5% in O₂ cost at submaximal intensity (80% VO_{2 max}) was observed. Subsequent studies using BR supplementation confirmed similar findings (Bailey et al. 2009, 2010; Vanhatalo et al. 2010; Cermak et al. 2012; Wylie et al. 2013a) to those observed by Larsen et al. (2007), although some positive changes have been also observed in VO_{2 max} (Larsen et al. 2010). To date, a direct effect of any form of NO₃⁻ supplementation on VO_{2 max} is elusive, and more studies are needed. However, NO₃⁻ supplementation benefits on exercise economy seem to be ineludible (Jones 2014b). On the other hand, supplementation of both BR and CIT separately has shown improvements in aerobic performance. Hoon et al. (2014) observed improvements in aerobic power, defined as time to complete a 2000 m test in highly trained rowers after ingesting 140 ml BR juice (8.4 mmol NO₃⁻) 2 h before exercise. Their results were later confirmed in a study carried out on trained cyclists, as the authors observed that BR supplementation 2.5 h before the exercise reduced the time necessary to complete the 4 km time trial (Lausch et al. 2017). Interestingly, in the study by Lausch et al. (2017), the BR supplementation was lower (6.2 mmol NO₃⁻) than in the Hoon et al. (2014) study. Beyond acute supplementation, NO₃⁻ chronic intake elicits potential interest due to the evidenced muscle’s capacity to accumulate, transfer and metabolize NO₃⁻ and nitrite (NO₂⁻), and its key role in whole-body NO pool maintenance (Nyakayiru et al. 2020; Piknova et al. 2022). In this regard, Cermak et al. (2012) and Rokkedal-Lausch et al. (2019) found improvements in 10-km cycling time trial in trained and well-trained cyclists after 7 days of supplementation with 12.4 and 8.8 mmol BR juice, respectively. Similar results have been reported following short-term chronic CIT supplementation. For instance, improvements in time to complete a 4 km cycling time trial were shown after ingesting 2.4 g of CIT for 7 days (Suzuki et al. 2016). Chronic intake (daily intake during 9 consecutive weeks) of 3 g/day of CIT combined with 2.1 g/day of BR extract led to improvements in estimated VO_{2 max} (using the following formula: 22.351 × Distance covered (km) in Cooper test – 11.288) and aerobic power performance of experienced triathletes when compared to placebo and BR extract conditions alone (Burgos et al. 2021). In the current study, the obtained peak power in the incremental aerobic exercise protocol test showed significant differences when comparing co-ingestion of CITG and BRG to PLAG, but we did not observe differences vs. the BRG condition. These benefits, however, were not accompanied by improved VO_{2 peak} or heart rate responses. Although more data are needed to confirm the effects observed in our study and to better understand the physiological mechanisms behind them, results might suggest that short-term chronic (i.e., 7 days) co-supplementation of CIT and BR supplements could improve peak power elicited during an aerobic performance test in elite male rowers.

Previous literature has described that NO may have a positive effect on blood vessel dilation and blood flow increment specifically in type II muscle fibers (Ferguson et al. 2013; Jones et al. 2016). BR juice can improve the release and re-uptake of calcium in the sarcoplasmic reticulum, thus improving muscle contractile capacities (Hernández et al. 2012). Therefore, NO₃⁻ supplements intake could potentially offer positive effects in high-intensity efforts where the ability to deliver and maintain high peak power is determinant, such as rowing (Bell et al. 1989). Wylie et al. (2013b) found, in recreational team-sport players, improvements in 6 s sprints after supplementing 8.4 mmol NO₃⁻ for 8 days, but no other benefits were reported in sprints lasting between 30 and 60 s. However, using similar supplementation (8 mmol NO₃⁻ for 7 days), benefits in 60 s sprint have been found in active men (Rowland et al. 2023). Although other benefits have been reported in efforts lasting 30–60 s such as after a Wingate test (i.e., 45 s), these studies used acute intake of NO₃⁻ (Dominguez et al. 2017b; Jodra et al. 2020). However, when combined supplementation with BR extract and CIT was given to triathletes for 9 weeks, improvements in 1 min maximal abdominal test were shown, suggesting potential synergic effects between both supplements (Burgos et al. 2021). CIT supplementation can stimulate muscle protein synthesis and, therefore, could potentially improve muscle strength (Goron et al. 2019). Nonetheless, the literature addressing the unbiased effect of CIT supplementation on sport performance is scarce. In our present study, the combined supplementation strategy showed no effect (Fig. 2) on 45 s Wingate test performance and its related outcomes (peak power, mean power, relative power, and fatigue index), suggesting 1 week of supplementation is not enough to generate hypothesized muscle adaptations. Interestingly, we showed a better blood lactate concentration clearance in the BR-CITG condition compared to BRG and PLAG conditions after 5 min post-Wingate test (Fig. 3). As lactate clearance relies on oxidative capacity, it is plausible that the reported effects of supplementation on aerobic metabolism could positively impact on athletes’ oxidative capacity and therefore be an indirect sign of improved aerobic performance. Some studies have also reported lower blood lactate concentrations after CIT supplementation (Rhim et al. 2020; Divito et al. 2022), but the literature regarding BR supplements remains unclear. In fact, potential mechanisms related to lower blood lactate values after CIT supplementation might be related to enhanced aerobic pathway, as reported for instance by higher levels of plasma lactate dehydrogenase enzyme (Rhim et al. 2020), which could be in accordance to previously identified effects of NO-related supplements such as BR. In addition, our study showed no improvements in CMJ jump between conditions (Table S1), a finding that contrasts with improvements found in horizontal jump after 9 weeks of the same combined supplementation (Burgos et al. 2021), and may suggest the need for longer than 7 days supplementation periods to improve strength and high peak power outcomes in elite rowers. Therefore, these results refuted the proposed hypothesis of a complementary way of improving muscle strength through a better excitation–contraction coupling after 6 g CIT and 3.5 g BR extract (500 mg or 8 mmol NO₃⁻) supplementation. As it has been hypothesized that elite athletes are potentially less sensitive to improvements after BR supplementation (Dominguez et al. 2017a), it is plausible that quantities required to show beneficial effects might be higher than 8 mmol NO₃⁻/day or should be related to body weight or fat-free mass, to individualize the supplement dose to the athlete.

Concerning biochemical biomarkers, we showed no differences between conditions after 1 week of supplement intake. Although some literature has studied the influence of CIT and BR supplementation alone on recovery status through hormones and functional tests (da Silva et al. 2017; Garnacho-Castaño et al. 2020; Daab et al. 2021), their effect remains unclear and more studies are needed. However, 9 weeks of BR + CIT supplementation showed improvements in the testosterone-to-cortisol ratio accompanied by enhanced aerobic performance test (Burgos et al. 2022). Considering all together, our study reinforces the idea to study longer periods of CIT and BR combined intake in elite rowers.

The impossibility to measure blood NO₃⁻ and NO₂⁻ concentrations in participants after ingesting supplements elicits a potential limitation that, however, it can be less determinant considering the chronic supplementation protocol used in this study. Methodologically, the absence of a CIT-only supplementation group can be considered a limitation to be noted, although CIT alone did not show any benefits compared to BR-only and BR + CIT supplementation in previous studies (Burgos et al. 2021, 2022). Moreover, muscle and strength-related effects could be taken as speculative as only performance tests were performed and no muscle samples (e.g., biopsies) were obtained for analyses. Finally, considering the impact of oral and gut microbiota and environment in NO₃⁻ and NO₂⁻ metabolism that can lead to different results (González-Soltero et al. 2020), the lack of rowers’ microbiota samples could represent a limitation. Nevertheless, the sample was composed of high-level rowers, which is a real strength of this study, due to the difficulty to access to this kind of athletes. Moreover, the design of the study and performance tests included in the study should be also acknowledged.

Future research should continue to study the combination of NO-related supplements in the long term to better understand their role in muscle NO₃⁻ and NO₂⁻ storage and related physiological and metabolic mechanisms. In addition, the potential synergic effects of supplements using compatible pathways in different performance outcomes could be of interest. Moreover, interventions lasting between 1 and 9 weeks should be addressed to be able to design higher efficacy supplementation protocols. Beyond measuring performance, mechanisms which by NO could impact oxidative and glycolytic metabolism, and muscle contractile function should be also studied in detail as this remains partially unknown yet. Finally, the impact of exogenously stimulated NO production in female athletes remains to be elicited to date, and therefore, it should be widely studied.

Combining supplements with similar physiological and metabolic mechanisms is interesting to optimize supplementation protocols in athletes regardless of their training/competition level. This study showed that even elite rowers find certain (although slightly) performance improvements by BR + CIT supplementation for 1 week.

The daily combination of 3.5 g of BR extract plus 6 g of CIT supplementation for 7 days seems to improve blood lactate clearance (oxidative capacity) compared to the PLA after the completion of a Wingate test, and the peak power after an aerobic performance test in a sample of elite rowers. No further improvements in maximal strength, high-intensity power and peak stroke power, and other aerobic performance-related outcomes were observed between supplements conditions. Our results may suggest that more than 7 days are necessary to better understand the potential synergic effects of BR + CIT supplements on sports performance.