Effects of chronic supplementation with beetroot juice on intermittent, high-intensity exercise efforts
Four of the studies reviewed tested the effects of taking beetroot juice supplements for 5 to 7 days on intermittent, high-intensity efforts [55, 56, 60] or on a resistance training session . Three of these studies detected a significant effect of beetroot juice supplementation [56, 58, 60] while in the remaining study, no significant difference compared with the placebo was noted .
Effects of chronic supplementation with beetroot juice on resistance training
Resistance training is used to improve muscular hypertrophy, strength, power and muscular endurance . Training sessions targeting muscle hypertrophy include workloads of around 70–85% 1 RM and 8–12 repetitions, while those aiming to improve muscular endurance include loads of around 50% 1 RM and some 15–25 repetitions . Such exercise sessions are largely dependent on glycolytic metabolism; the lactate threshold in resistance training exercises such as half squat is detected at ~25% 1 RM [63, 64]. To determine the effects of 6 days of beetroot juice supplementation (6.4 mmol NO3) on resistance training sessions designed to improve local muscular hypertrophy and endurance, in the study by Mosher et al. reviewed here , the number of bench press repetitions accomplished in three sets using loads equivalent to 60% 1 RM was recorded. Results indicated that supplementation increased the number of repetitions in the three exercise sets improving session performance by 18.9%.
In an earlier investigation, the effects of sodium bicarbonate supplements were assessed in a similar study to the one by Mosher et al. . Subjects performed 3 sets until exhaustion with loads of 10–12 RM in three exercises targeting the lower limbs . Results indicated that, like the beetroot juice, sodium bicarbonate supplementation led to more repetitions in the session . However, in parallel with the increasing number of repetitions, blood lactate concentrations also rose (~2.5 mmol) . This was not observed in Mosher’s study .
If we consider the nature of resistance training, the athlete passes from a resting condition to a situation demanding high energy levels during the first repetitions of a set. Because the phosphagen system is the main energy pathway in rest-exercise transitions , phosphocreatine reserves may be depleted in response to a resistance training exercise set. Recovering these reserves takes some 3–5 min . Given that phosphocreatine resynthesis is dependent on oxidative metabolism  and that beetroot juice has an ergogenic effect on exercise modalities with a major oxidative metabolism component , it could be that this supplement accelerated this recovery during the rest period in Mosher’s study (2 min) and thus avoided progressive phosphocreatine depletion throughout the session. In turn, this faster rate of resynthesis would attenuate the increasing levels of adenosine diphosphate (ADP) and inorganic phosphates . Both these metabolites have been associated with the appearance of muscular fatigue . Hence, by delaying the build-up of critical levels of these metabolites, the appearance of fatigue will be delayed and this will allow for more repetitions in sets until exhaustion . NO3− supplementation could also improve muscle efficiency and contractile capacity by promoting the release of calcium from the sarcoplasmic reticulum in the muscle cells and its reuptake [52, 69]. Thus, a train of action potentials leading to an increased supply of calcium to the muscle fibre will increase the strength of muscle contraction .
Effects of chronic supplementation with beetroot juice on intermittent high-intensity exercise efforts
Some sport modalities such as team, racket or combat sports require bursts of high-intensity efforts followed by rest periods. Thus, in team sports, high-intensity efforts (~3–4 s) are interspersed with variable active rest periods . In racket sports like tennis, efforts last 7–10 s and rest periods 10–16 s (between points) and/or 60–90 s (side changes) . Finally, in combat sports more intense efforts are 15–30 s long and active rest periods are 5–10 s long every 5 min . In all these sports modalities, the capacity to repeat high-intensity efforts with only short recovery periods is considered a performance indicator . This means that higher level athletes are able to maintain performance in successive high-intensity intervals over a long time period .
To find out if beetroot juice supplementation would improve this ability to repeat high-intensity efforts during a team sport match, Thompson et al.  administered beetroot juice over 7 days to a group of athletes (12.8 mmol NO3−). The performance test consisted of two blocks of five 6-s sets of sprints on a cycle ergometer with 14-s active recovery periods in the middle and end of a simulated match lasting 2 × 40 min . The results of this study indicated a total work volume improved by 3.5% in the whole session, though this improvement was greater at the end of the first half (at half time).
If we again consider the nature of this type of exercise, it has been established that it involves the recruitment of type II muscle fibres [75, 76], which are more powerful though show more fatigue than type I units . This lesser resistance to fatigue has been related to reduced blood flow and myoglobin concentrations in these muscle fibres compared to type I. Hence, type II muscle fibres are designed to promote non oxidative pathways and have shown a greater creatine storage capacity  for an enhanced metabolism of phosphocreatine  and proteins with a buffering effect at the intracellular level such as carnosine , favouring a glycolytic type metabolism.
Animal studies have shown that increased blood flow in response to NO3− supplementation is greater in type II compared to type I muscle fibres . This greater irrigation and oxygen availability in the recovery period along with a greater creatine storage capacity of motor type II units  (promoting phosphocreatine resynthesis ) means that during an exercise effort followed by a short rest period (14 s), beetroot juice supplementation could delay phosphocreatine depletion during successive sprints and explain the improvements noted by Thompson et al. .
Despite such greater effects of NO3− supplementation on type II versus type I muscle fibres, animal studies have also shown that effects on calcium release and reuptake in the muscle cell sarcoplasmic reticulum is greater in type II than type I muscle fibres . Accordingly, because of the important role of type II muscle fibres during sprints [75, 76], supplementation could have led to an improved capacity to generate muscle power and thus explain the significant improvements in performance observed by Thompson’s group.
Buck et al.  examined the effects of 6 days of supplementation with beetroot juice (6.4 mmol NO3−) or sodium phosphate (50 mg·kg lean mass) on performance in a test consisting of repeated sprints as 6 sets of 20 m and 25-s of rest between sets in the middle and end of a simulated match lasting 60 min. The beetroot juice intervention did not improve performance at these sprints, yet did do so when taken along with sodium phosphate (2%) compared with placebo, though this improvement was of lesser magnitude than when the subjects only took sodium phosphate supplements (5%). These findings suggest that, unlike beetroot juice, sodium phosphate intake may have an ergogenic effect in this protocol. If we compare the tests used by Buck et al.  and Thompson et al. , work periods were shorter (2–3 vs 6 s), while rest periods were longer (25 vs 14 s). Therefore it could be that 2–3 s efforts lead to a significantly lower reduction of phosphocreatine reserves at the end of these efforts. Further, the 25 s of rest approaching the 30 s in which the recovery of 50% of phosphocreatine stores takes place , may have been sufficient to stabilize reserves of phosphocreatine and therefore avoid the appearance of fatigue .
Another study investigated the effects of longer term supplementation (5 days) with beetroot juice (8.4 mmol NO3−), this time on performance in a repeated high-intensity test . These authors sought to determine supplementation effects on different exercise protocols. Subjects performed a session consisting of twenty four 6-s sets of work and 24 s of rest between sets, a second session of two 30-s sets of work and 2 min of rest between sets and a third session of six 6-s sets and 60 s of rest between sets. As did Thompson et al. , Wylie et al.  selected 6-s exercise sets in the first session though rest intervals were longer (24 vs 14 s). Another difference was that the participants had not first undergone fatigue (in the simulated team sport match) before the performance test. Notwithstanding, results were similar in that mean power generated in the sets over a whole session improved by ~7%. However, improvements across the 24 × 6–24 protocol were not comparable to those recorded in the other two tests, in which no significant improvements were recorded.
In the test protocols including 30-s and 60-s work efforts, beetroot juice supplementation resulted in no improvements in any indicators of performance . These protocols consisting of longer duration work intervals mainly involve a glycolytic type metabolism and in smaller measure elicit the high-energy phosphagen system. An increase in glycolysis leads to increased H+ production, lowering pH . To avoid increasing acidosis, a series of responses targeted at reducing phosphofructokinase take place including diminished glycolysis  and phosphocreatine resynthesis , and muscle contractibility modifications . Such responses manifest as reduced non aerobic metabolism or a reduced capacity for muscle power and strength, in other words, fatigue . Supplements such as β-alanine (which increases muscle carnosine concentrations , a protein that acts as a buffer inside the cell ) and sodium bicarbonate  (main extracellular buffering agent) have shown ergogenic effects on performance at high-intensity efforts involving the predominance of glycolytic metabolism . The combined effect of these supplements is greater than the impact of each supplement on its own .
Although beetroot juice supplementation induces vasodilation and increased blood flow (in type II muscle fibres, recruited mainly in exercise bouts of 30 to 60 s duration), increasing available oxygen in the muscles, rather than being activated because of a lack of oxygen (anaerobiosis), non-oxygen dependent pathways are activated because of a greater demand for energy production via oxidative phosphorylation. Thus, these effects, although they potentiate oxidative phosphorylation, have no repercussions on glycolytic energy metabolism. Hence, as beetroot juice has no alkalizing effect supplementation with this product is unable to reduce acidosis, as the main factor limiting performance at efforts lasting 30–60 s. However, potentiating effects on aerobic metabolism increases the speed of phosphocreatine resynthesis, dependent on oxidative phosphorylation. This means it may be effective for repeated high-intensity efforts whose duration is close to 6–10 s, in which high energy phosphagens contribute mainly to the metabolism  and the work volume is sufficient to cause significant depletion, which when faced with short rest intervals leads to progressive depletion and consequently to fatigue. Accordingly, beetroot juice supplements can have an ergogenic effect when exercise efforts are intermittent, maximum intensity, short-duration (6–10 s) and interspersed with brief recovery periods (<30 s).
Effects of acute beetroot juice supplementation on intermittent high-intensity efforts
Five of the studies reviewed here were designed to analyze the effects of a single beetroot juice supplement on intermittent high-intensity exercise efforts [38, 53, 54, 57, 59]. Aucouturier et al.  administered the supplement (~10.9 mmol NO3−) to a group of recreation athletes 180 min before performing sets until exhaustion consisting of 15 s of pedalling at 170% VO2max followed by 30-s rest periods. The authors reported that the beetroot supplement gave rise to improvements close to 20% in the number of repetitions performed and the total work completed in the session . Besides the number of sets completed and the work accomplished, these authors measured red blood cell concentrations at the microvascular level. The beetroot juice, apart from improving performance, was found to increase microvascularization. Such improvements are considered a beneficial effect on oxygen exchange in the muscle . Accordingly, these oxygen availability improvements produced at the muscular level could have potentiated oxidative phosphorylation during rest periods, and, given their brief duration, could have increased phosphocreatine resynthesis when subjects took the supplement rather than the placebo. Thus, supplementation would have delayed the depletion of phosphocreatine reserves and this effect was likely the cause of the improvements observed in the repeated sets of intermittent sprints [94, 95].
As did Aucouturier et al. , Muggeridge et al.  examined the effect of beetroot juice (5 mmol NO3−) taken 180 min before an intermittent effort consisting of 5 sets of 10 s in a kayak ergometer with 50-s interset rest periods. In this study, though supplementation seemed to have a greater effect on the power generated in the last two sets, the improvement noted lacked significance. However, if we compare this study with the study by Aucouturier et al. , work periods in the Muggeridge study  were shorter (10 vs 15 s) and rest periods were much longer (50 vs 30 s). Ten second maximum intensity intervals have a significantly reduced capacity compared with 15s intervals to deplete phosphocreatine reserves. Moreover, the rate of phosphocreatine replacement has a first phase in which up to 50% of these reserves can be replenished in 30 s and 100% in 3–5 min . Also if we consider that the main effect of beetroot juice supplements is linked to an improved rate of phosphocreatine resynthesis, it is possible that as there is less depletion and a rest period in which there is almost complete recovery of phosphocreatine reserves, supplementation could not have exerted any beneficial effect in the study by Muggeridge et al. . However, despite the short work periods and relatively long recovery periods and the fact that the power developed in the last sets showed an improved trend following supplementation, it is possible that lengthening intervals in a set until exhaustion would have been beneficial and given rise to similar results to those observed by Aucouturier et al. .
Rimer et al.  assessed the effects of acute supplementation (150 min before exercise) with beetroot juice (11.2 mmol NO3−) on performance in a maximal intensity 3-s test on an isoinertial cycle ergometer and a 30-s test on an isokinetic cycle ergometer. Supplementation was effective at improving pedalling cadence, and thus the power generated, in the 3-s test. However, no such effect was observed in the isokinetic test.
The improvements noted by Rimer’s group in the 3-s test affected pedalling cadence. Because of the link between such improvements and an increase in muscle shortening velocity  and the proposal that NO could increase this velocity [97, 98], the authors suggested that beetroot juice could have a beneficial effect on power output . This rationale was also used to explain the lack of changes produced in the 30-s test in which pedalling cadence was fixed at 120 rpm. This means that any improved power production in the isokinetic test could only occur if there was an increase in power at a constant shortening velocity , since power equals force times velocity.
In a later investigation performed in CrossFit athletes, it was reported that supplementation with NO3− salts (8 mmol NO3) rather than beetroot juice was able to improve performance in a 30-s cycle ergometry test . However, unlike the 30-s test used by Rimer et al. , the test was isoinertial. The difference between the 2 cycle ergometers is that while in the isokinetic test pedalling cadence is prefixed and improvements only in strength are possible, in an isoinertial test the workload is fixed and any power improvements produced manifest as improvements in pedalling cadence. Given that beetroot juice supplementation could improve power development as a consequence of a reduced muscle shortening velocity [59, 97, 98], the isokinetic cycle ergometer is perhaps not sufficiently sensitive to assess the effects of this supplementation. Considering the beneficial effects on cadence and power output observed in the cycle ergometry 3-s  and 30-s  tests, it seems that beetroot juice supplementation could have a beneficial effect on this type of effort.
In a fourth study, Clifford et al.  assessed the effects of a single intake of beetroot juice on performance in a test of 20 sets of 30 m sprints interspersed with 30-s rest periods. These authors observed no ergogenic effects of the supplementation. However, if we look at the characteristics of the test employed by the researchers, we find that the work periods (close to 3 s) together with the 30 s recovery periods could be sufficient for the subjects to have recovered their phosphocreatine levels in the rest intervals, minimizing the possible ergogenic effects of the supplementation.
A novel indicator used in this study by Clifford et al.  was the counter-movement jump (CMJ) test performed before the intermittent velocity test and in the rest periods. Performance in this test is determined by the contractile properties of muscle and by neuromuscular control of the entire musculoskeletal system . Given that fatigue reflects the incapacity of the neuromuscular system to maintain the level of power required , losses in CMJ height at the end of exercise are taken as an indicator of muscular fatigue .
In the study by Clifford’s group , it was observed that the protocol of intermittent sprints gave rise to muscular fatigue. This fatigue can be the outcome of deficiencies in the muscle’s contractile mechanism [101, 103]. Alternatively, strong eccentric actions of the hamstring muscles during sprints may produce muscle damage  and therefore modify the structure of the muscle fibre’s sarcomeres. Thus, any loss in CMJ height could indicate muscle damage. While CMJ was monitored after the protocol of 20 sets of 30 m with 30-s rest periods, a greater recovery of CMJ height was observed in the supplementation group. This suggests that beetroot juice could help preserve muscle structure during high-intensity efforts. Another explanation could be related to the vasodilation effect of beetroot juice  possibly helping muscle regeneration during early recovery. In future work, biomarkers of muscle damage or inflammation need to be examined.
In the fifth study, Martin et al. investigated the effects of beetroot juice (6.4 mmol NO3−) on repetitive sets until exhaustion each consisting of 8 s of work followed by 30 s of rest on a cycle ergometer . No effects were detected on power output in the different sets. Moreover, a lower number of sets was accomplished in the session for the supplementation group versus placebo group. In effect, this was the only study to describe an ergolytic effect of beetroot juice. The authors argued that because of the scarce contribution of oxidative phosphorylation to energy metabolism during high-intensity efforts and that the ergogenic potential of this supplement is related to potentiating oxidative pathways, no beneficial effects are produced on this type of physical action.
The results of the investigation by Martin et al.  conflict with those of others who did observe beneficial effects on performance in similar tests [54, 56, 58, 60]. Beetroot juice was taken 120 min before exercise. This regimen is not appropriate, as peak NO2− levels are produced 2–3 h after ingestion and it is recommended that supplementation should be taken at least 150 min–180 min before the high-intensity effort [32, 50]. Effectively, Aucouturier et al.  used a test of similar characteristics but the beetroot supplement was taken 180 min before the exercises, as recommended.