Introduction
Nitric Oxide (NO) is an influential signaling molecule, which rapidly acts on vascular smooth muscle via guanylate cyclase (18). The result of this causes increases in cyclic guanosine mono-phosphate, which facilitates relaxation effects in smooth muscle, further producing vascular dilatation (13). Dietary nitrate (NO3−) typically obtained from green leafy vegetables and in particular from beetroot is reduced to NO2− by nitrate reductase (31), causing a sustained increase in circulating NO2− levels (23). It is principally a vasodilator, producing a relaxation effect upon the vascular endothelium (19). Dietary NO3− is 100% bio-available (14,21) and is readily absorbed in the upper gastrointestinal tract (28). Dietary nitrate and nitrite can therefore be considered storage pools for NO bioactivity (14,23).
Dietary supplementation with either sodium nitrate or nitrate-rich beetroot juice (BR) has consistently been shown to improve exercise time to exhaustion during both endurance running (20) and cycling (6). It has also been shown to improve cycling time trial (11) and repeated intermittent rowing performance (9,12,20) and improving exercise tolerance at altitude (27). Such improvements in performance have typically ranged from between 1.2 and 2.9%, but some recent studies with highly trained participants have observed no improvement in performance after the acute single-dose ingestion of BR (12,29). However, a relatively common finding in studies examining the ergogenic effects of nitrate on endurance exercise performance is that heart rate, lactate concentration, CO2 production, minute ventilation, and respiratory-exchange ratio all show no significant change between nitrate and placebo groups.
There have been only 4 studies (4,17,22,26) that have investigated the effects of BR supplementation or betaine (an amino acid constituent in beetroot jouice) on resistance training. Results regarding the use of betaine are equivocal, but suggest a modest increase in resistance exercise performance. Bailey et al. (4) recruited 7 recreationally active men that consumed either 0.5 L·d−1 of BR (5.1 mmol·d−1 NO3−) or a placebo for 6 days. During the last 3 days of supplementation, participants completed low-intensity and high-intensity (15 and 30% maximal voluntary isometric contractions, respectively) “step” knee extension tests. Their data showed that BR increased plasma nitrite concentrations by 240% and decreased pulmonary V̇o2 by 25% from rest to low-intensity exercise. Beetroot juice consumption also reduced by the amount of phosphocreatine (PCr) degraded during low-intensity exercise and high-intensity exercise by 36 and 59%, respectively, compared with placebo. Reductions in PCr usage were also accompanied by a reduced total ATP utilization during both high- and low-intensity exercise, suggesting potentially ergogenic effects for this type of exercise. This is the first study that has investigated the effects of a prolonged dose of BR before resistance exercise. Therefore, the aim of the present study was to investigate the effects of nitrate supplementation on the time to fatigue and subjective ratings of exertion during resistance exercise performance and recovery.
Methods
Experimental Approach to the Problem
This experimental paradigm was based on the use of a beetroot juice supplement as an ergogenic aid to improve performance of recreational, weight training athletes in terms of fatigue. We decided to measure this by using the number of repetitions until fatigue without increasing physiological as measured by blood lactate or psychological fatigue as measured by RPE indicators.
Subjects
Twelve recreationally active men (age, 21 ± SD 2 years, height, 177.2 ± 4.0 cm, weight, 82.5 ± 9.8 kg) were recruited for this study. All participants were familiar with performing resistance exercises and had a minimum of 3 years training experience (4.2 ± 0.9 years) and a minimum weekly training frequency of 3 days a week (4 ± 1 day), and 3 hours per week (6 ± 2 hours). Written informed consent was obtained from all participants before completing health screening for contraindications to exercise. Participants were required to abstain from ingesting ergogenic supplements during the data collection period. Furthermore, they were also required to replicate dietary intakes for the 24 hours before each laboratory visit, avoid exhaustive exercise (9), and limit caffeine consumption (15). Before each laboratory visit, the participants were instructed to eat no less than 3 hours before the trials and consume only water in the final hour before testing commenced (9). Participants were requested to continue their normal dietary intake during the study and this was verbally checked with each individual before testing. The study was approved by the Departmental Research Ethics Committee. The study conforms to the Code of Ethics of the World Medical Association (approved by the ethics advisory board of Edge Hill University) and required players to provide informed consent before participation.
Procedures
The initial laboratory visit required each participant's bench press one repetition maximum (1RM) to be determined using a previously validated protocol (3). Participants completed a warm-up set of 12 repetitions at 10% of estimated 1RM, which was performed using a Smith machine (Hammer Strength; Life Fitness, Ely, United Kingdom) followed by a 1-minute rest period. The load was then increased by 20% and a set of 6 repetitions was then completed followed by a 2-minute rest period. A near maximal load was then estimated, which was loaded onto the equipment for the participant to complete 3 repetitions, followed by a 2–3 minute rest period. After these initial warm-up sets, the participants estimated 1RM was established and one attempt was made followed by a 2–4 minutes rest. If completed, 1.25, 2.5, or 5 kg was progressively loaded to attempt to find the actual 1RM. The 1RM was obtained within 3 to 6 sets for all participants.
After the establishment of 1RM, participants attended the laboratory on 2 further occasions. The study used a double-blind, randomized cross-over design requiring participants to complete a 6-day ingestion period of either a 70-ml BEET It Sport nitrate shot or a blackcurrant placebo drink (6). The nitrate shot contained 6.4 mmol·L−1 or 400 mg of nitrate (2). The order in which participants consumed the nitrate or placebo was administered by a laboratory technician, and this order was only divulged to investigators on completion of all of the data collection. The supplementation strategy required participants to ingest either a 400 mg nitrate shot or placebo on each of the 6 consecutive days. No participants reported any adverse gastrointestinal discomfort during either of the ingestion periods. Two experimental trials were then performed, one for each of the ingestion strategies, which were separated by a minimum washout period of 72 hours, a strategy that has previously been used in a study with a similar design (30). These trials took place on the last day of ingestion at the same time of day (10). The experimental trials consisted of a warm-up of 2 sets of 12 repetitions of bench press. The first set was performed at 10% and the second at 20% of established 1RM using the Smith machine. This was followed by a 1-minute rest period that took place after each warm-up set. Testing procedures then began performing sets of repetitions to failure using a load of 60% of 1RM, with each set separated by a 2-minute rest period (8,15). Number of repetitions, total weight lifted, and local ratings of perceived exertion (RPE) were recorded on completion of each set using 1–10 scales where 1 represented no exertion at all and 10 being maximal exertion. Pre-exercise and postexercise blood lactate was measured from capillary blood samples (Micro-stat P-GM7; Analox, London, UK).
Statistical Analyses
All data were assessed for normality using standard graphical methods. Between-condition analysis for all variables measured after each set were analyzed using repeated measures ANOVA. Violations to sphericity were determined using a Mauchley's test. If a significant violation was observed, significance was adjusted using either the Huynh-Feldt or Greenhouse Geisser correction technique, where sphericity >0.75 and <0.75, respectively. Post hoc analysis was conducted using the Bonferroni pairwise comparison. Total weight lifted and total number of repetitions were analyzed using paired t-tests for both condition effects and order effects. Statistical significance was set to p ≤ 0.05 for all tests. Calculations of effect sizes were done using ηp2 for ANOVA and Cohen's d for t-tests. All data were analyzed using SPSS v22 (IBM, Portsmouth, United Kingdom).
Results
There was a significant improvement in the total number of repetitions in all 3 sets (Figure 1) in the nitrate trial compared with the placebo (F = 29.62, p < 0.001, ηp2 = 0.70). This resulted in significantly greater total repetitions completed (mean difference = 6.92, t = 5.44, p < 0.001, d = 0.96) and total weight lifted (mean difference = 411.3 kg, t = 5.00, p < 0.001, d = 0.52) in the nitrate trials (Figure 1 and Table 1, respectively). No order effect on resistance exercise performance was observed because neither total weight lifted nor total repetitions differed between trial 1 and 2 (mean difference = 56.7 kg, t = −0.41, p = 0.69, d = 0.16; and mean difference = 1.6, t = −0.73, p = 0.48, d = 0.29, respectively). Despite the observed increases in resistance exercise performance in the nitrate condition, there were no significant differences in the lactate responses (Table 1) between the trials (F = 1.79, p = 0.20, ηp2 = 0.12), but in both experimental conditions the performance of the bench press protocols did cause significant increases in lactate concentrations (f = 306.68, p < 0.001, ηp2 = 0.96), without an observed condition × time interaction (F = 4.31, p = 0.058, ηp2 = 0.25). Interestingly, despite increases in resistance exercise performance in the nitrate condition, there were no observed differences in either RPE or RPE-L between conditions (F = 0.69, p = 0.42, ηp2 = 0.05) and (F = 0.06, p = 0.81, ηp2 = 0.01, respectively) although there were significant increases in RPE-L between each of the sets (F = 24.09, p < 0.001, ηp2 = 0.65).
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Figure 1.: Mean (±SD) number of bench press repetitions for each set and condition totals. (*) denotes a significant main effect for condition (p ≤ 0.05).
Table 1.: Mean (±SD) physiological and perceived exertional responses.
Discussion
This study investigated the effects of 6 days of nitrate supplementation in the form of beetroot juice on resistance exercise performance. It was hypothesized that nitrate would increase the number of repetitions until fatigue without increasing physiological or psychological fatigue indicators. The principle findings of this study, the first of its kind, established that total work increased in terms of total weight lifted and repetitions until failure at 60% of 1RM. Both improvements occurred without significantly affecting physiological responses in terms of blood lactate concentration and psychological fatigue indicators of local and general rate of perceived exertion.
Nitrate in the form of beetroot juice has been shown to increase plasma nitrite (6,21) and these increases have been identified as a significant factor in influencing exercise tolerance (24). This has been investigated by Bailey et al. (4) providing substantial evidence that increased plasma levels of nitrate are associated with a 25% increase in intermittent exercise performance during a 2-legged knee extensor exercise protocol. The current study found similar percentage increases in resistance exercise performance to failure with a mean average of 19.4% in total repetitions and mean average of 18.9% in total weight lifted. Although blood plasma nitrite levels were not taken within the current study, it is presumed that plasma levels increased because of the reports from previous studies and increased performance within this study. The increased tolerance is a result of increased energy yield through PC stores (7), which is supported by findings from the work of Bailey et al. (4). They established that with high-intensity exercise, specifically leg extension exercise, there was a reduction in PC degradation and a decrease in the production of Adenosine di-phosphates and inorganic phosphates, which are metabolites that have been linked to fatigue within muscles (8). This work concludes that nitrate supplementation could provide a more sufficient rate of change between these substrates and metabolites, consequently delaying the time until they reach critical values. This in turn enables a greater time until depletion which results in a more efficient usage of PC stores at high-intensity exercise, prolonging the tolerance at the given work rate. In relation to the current study findings, PC stores were used more efficiently when performing the repetitions until fatigue within a given set, allowing individuals to sustain muscle contractile performance that last longer. This could explain the increase in total repetitions, more specifically increases in repetitions within each set owing to the nitrate ingestion.
Dietary nitrate supplementation has also been reported to excitation-contraction coupling and efficiency (5,16). These responses occur through an increase in sarcoplasmic reticulum calcium release and increase in force production per power stroke within type 2 fibers (16). The excitation-contraction coupling occurs when signals at the surface of the cell couple to cause releases in calcium within the sarcoplasmic reticulum; consequently, this induces actin–myosin interactions resulting in muscle fiber contraction (1). The calcium is then withdrawn by the ATP-dependent pump (25). When multiple burst action potentials occur, calcium is sustained and greater contraction force can occur (25). These components modulate the effectiveness and efficiency of the force that is produced by this process. Greater NO production has been proposed to enhance the efficiency of coupling between the above components to produce this overall effect of greater muscle contractile function (5,25). Hypothesizing that these given effects are apparent in humans (4), these enhancements in muscle efficiency within type 2 fibers could result in lower overall energy turnover (5). The above evidence suggests, for our work, that when participants were completing upper body (bench press) exercise until fatigue, more effective muscle contraction was apparent with lower overall energy cost, meaning the nitrate aided in conserving energy over the given sets, thus allowing participants to complete more sets.
Practical Applications
Coaches and their athletes alike could make use of nitrate supplementation because our evidence suggests that it can be used within healthy male resistance-trained population to increase exercise tolerance and improve performance. Further studies are necessary to investigate long-term use and possible adaptations to resistance training with longer periods of dosing with nitrate.
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