Dietary Nitrate Supplementation and Exercise-Related Performance

Abstract

Over the last decade, there has been a growing interest in the utility of nitrate (NO3⁻) supplementation to improve exercise-related performance. After consumption, dietary NO3⁻ can be reduced to nitric oxide, a free radical gas involved in numerous physiological actions including blood vessel vasodilation, mitochondrial respiration, and skeletal muscle contractile function. Emerging evidence indicates that dietary NO3⁻ supplementation has a small but nevertheless significant beneficial effect on endurance performance through the combined effects of enhanced tissue oxygenation and metabolic efficiency in active skeletal muscle. There is further evidence to suggest that dietary NO3⁻ exerts a direct influence on contractile mechanisms within the skeletal muscle through alterations in calcium availability and sensitivity. Response heterogeneity and sizeable variability in the nitrate content of beetroot juice products influence the effectiveness of dietary NO3⁻ for exercise performance, and so dosing and product quality, as well as training history, sex, and individual-specific characteristics, should be considered.

Over the last decade, there has been growing interest in the use of dietary nitrate (${\text{NO}}_3^{-}$) supplementation to improve sport performance in endurance and power-dependent disciplines. Endurance events, characterized by rhythmic contractions of large muscle groups lasting longer than 2 minutes in duration (eg, 800-m run, 200-m swim), necessitate ample distribution and utilization of oxygen to active skeletal muscle tissue.¹ Success in power-dependent events, on the other hand, is primarily contingent upon generating maximal force as quickly as possible (eg, Olympic weightlifting or shot put).

${\text{NO}}_3^{-}$ is a precursor to nitric oxide (NO), a free-radical gas involved in numerous physiological processes, including blood vessel vasodilation, mitochondrial respiration, and skeletal muscle contractile function.² Endogenous synthesis of NO occurs continuously within the vascular endothelium as well as in the tissues themselves via the L-arginine pathway.³ However, NO can also be produced through the reduction of ${\text{NO}}_3^{-}$ to nitrite (${\text{NO}}_2^{-}$), especially during situations of high-intensity physical exertion when the need for oxygenated blood to active tissue increases.⁴ As exercise intensity rises, a greater amount of oxygen is consumed by contracting skeletal muscles—resulting in an increased number of metabolic by-products (eg, hydrogen ions). With repeated contractions, decreasing oxygen tension and pH level in the skeletal muscle microenvironment further accelerate the transition of ${\text{NO}}_2^{-}$ (from dietary ${\text{NO}}_3^{-}$) to bioavailable NO.⁵

${\text{NO}}_3^{-}-{\text{NO}}_2^{-}-\text{NO}$ Pathway and Exercise

The reduction of inorganic ${\text{NO}}_3^{-}$ found in dietary sources (eg, beetroot, spinach) to NO occurs in parallel with the endogenous synthesis of NO from L-arginine. When consumed, ${\text{NO}}_3^{-}$ is concentrated in saliva and becomes rapidly reduced to ${\text{NO}}_2^{-}$ by anaerobic bacteria in the oral cavity.⁶ ${\text{NO}}_2^{-}$ is then reduced to NO as digestion continues by the acidic environment of the stomach and is absorbed along with remaining ${\text{NO}}_2^{-}$ into the plasma from the upper gastrointestinal tract.^5,7,8 ${\text{NO}}_2^{-}$ can be further reduced to NO during situations of increased metabolic demand (ie, physical exertion) when contracting skeletal muscles extract a greater amount of oxygen from the peripheral blood per unit time.^4,5 In comparison with resting or sedentary conditions, a heightened level of NO is available to elicit effects on blood vessels and skeletal muscles during exercise.⁹ Figure 1 illustrates the pathway by which exercise influences the reduction of ${\text{NO}}_3^{-}$ to NO.

FIGURE 1.

The influence of exercise on the ${\text{NO}}_3^{-}-{\text{NO}}_2^{-}-\text{NO}$ pathway after the ingestion of dietary ${\text{NO}}_3^{-}$. Conditions within exercising skeletal muscle, namely, a relatively lower pH and greater extraction of oxygen from the peripheral blood, promote the reduction of ${\text{NO}}_2^{-}$ to NO.

The diverse role of NO in the regulation of blood flow, oxygen uptake, and skeletal muscle contractions has implications for athletic performance.^2,10,11 Accordingly, dietary ${\text{NO}}_3^{-}$ supplementation has been shown to result in small but nevertheless significant improvements in the performance of specific endurance and power-dependent disciplines in high-quality, placebo-controlled, double-blinded studies. However, the extent to which dietary ${\text{NO}}_3^{-}$ ingestion may influence performance is varied and likely dependent on factors involving the dose of ${\text{NO}}_3^{-}$ consumed, as well as training history, sex, and other individual-specific characteristics.

Dietary ${\text{NO}}_3^{-}$ Dosing Strategies and Supplementation for Performance Enhancement

Although a variety of leafy green and root vegetables contain ${\text{NO}}_3^{-}$, most studies evaluating the influence of dietary ${\text{NO}}_3^{-}$ supplementation on exercise performance have used bottled beetroot juice beverages.¹² Concentrated forms of dietary ${\text{NO}}_3^{-}$, like beetroot juice, deliver more ${\text{NO}}_3^{-}$ per volume, making it an attractive option for ingestion before athletic pursuits. For example, an athlete would need to consume 12 cups of raw spinach to ingest the equivalent amount of ${\text{NO}}_3^{-}$ provided by a single 140 mL (≈2/3 cup) serving of commercially available beetroot juice.^13,14

To date, recommendations for the minimum amount of dietary ${\text{NO}}_3^{-}$ needed to improve exercise performance are lacking. Doses of dietary ${\text{NO}}_3^{-}$ ranging from 3.2 to 10.4 mg·kg body mass⁻¹ taken either acutely (2–3 hours before exercise to coincide with peak plasma ${\text{NO}}_2^{-}$ and ${\text{NO}}_3^{-}$ levels) or chronically (over several days to weeks) have shown improvements across endurance, power-dependent, and high-intensity intermittent exercise performance (Table). A minimally effective dose of 527 mg—equivalent to 7.5 mg· kg body mass⁻¹ for a 70-kg individual—has been suggested.² However, it is worth noting that the ${\text{NO}}_3^{-}$ content of commercially available beetroot juice is highly variable and is rarely independently tested to confirm amounts provided by the manufacturer. For example, Gallardo and Coggan¹⁴ found a ≈50-fold range in ${\text{NO}}_3^{-}$ content among 24 different beetroot juice products marketed to athletes. Of note, only 2 of the products tested consistently provided an amount of nitrate greater than the minimum dose (527 mg) suggested to enhance exercise performance in most individuals.^2,14 As such, it is recommended that research studies evaluating the effects of beetroot juice supplementation on athletic performance use products of a known quality and dose and investigators should further verify that the ${\text{NO}}_3^{-}$ content of the product is equivalent to the amount provided by the manufacturer through independent laboratory testing.

TABLE.

Doses of Dietary ${\text{NO}}_3^{-}$ Resulting in Improved Exercise-Related Performance Taken Within 24 Hours Before Exercise

	Subject Characteristics	${\text{NO}}_3^{-}$ Dose (≈mg·kg Body Mass)
Endurance time-trial performance in events lasting 5–30 min	Combined15–21	6.9 (range, 3.2–10.4)
	Endurance trained men15–20	6.9 (range, 3.2–10.4)
	Recreationally active men and women21	6.9
Maximal power during single leg extension, cycling, and running exercise	Combined22–28	6.5 (range, 4.5–9.8)
	Resistance trained men or men with previous experience in power-dependent sports (ie, football, CrossFit)22,24,26–28	5.1 (range, 4.5–6)
	Mixed sports (ie, triathlon, tennis) men and women23	9.4
	Healthy men and women25	9.8
Intermittent high-intensity exercise performance	Combined22,29–33	6.7 (range, 4.5–10.4)
	Team-sports players men22,29–33	6.7 (range, 4.5–10.4)

Furthermore, most studies evaluating the effects of dietary ${\text{NO}}_3^{-}$ on athletic performance have not accounted for variations in baseline subject diet. For example, although most interventional studies require that subjects maintain their habitual diet for the duration of the intervention period and ask that they avoid foods containing high amounts of dietary nitrate (eg, leafy green vegetables, rhubarb), often, the amount of additional dietary nitrate consumed is not quantified through dietary recall. As such, it is generally unknown whether the minimally effective dose for performance enhancement is higher than the estimated 527 mg.

Dietary ${\text{NO}}_3^{-}$ Supplementation and Endurance Performance

Substantial evidence from placebo-controlled double-blinded studies support the use of dietary ${\text{NO}}_3^{-}$ supplementation for improving aspects related to the performance of endurance events (eg, cycling, running, and rowing).^15,34–43 Because of its role as a potent vasodilator, NO increases blood flow (ie, tissue perfusion), which in turn supports oxygen delivery to metabolically active skeletal muscles.¹² Additionally, it has been suggested that NO increases skeletal muscle contractile function—indicating that more muscular work can be performed per unit time for the same metabolic cost (Figure 2).^{2,34,36,37,44} Reductions in the metabolic cost^{15,35–38,42,43} and the perception of effort³⁹ during continuous submaximal and maximal aerobic exercise have occurred with dietary ${\text{NO}}_3^{-}$ supplementation, presumably as a consequence of improved tissue oxygenation and contractile function. As such, individuals have demonstrated a heightened tolerance to high-intensity aerobic exercise after dietary ${\text{NO}}_3^{-}$ supplementation.^{34,35,39–41} Bailey et al³⁵ and Lansley et al⁴⁰ reported that recreationally active men cycled +15 to +16% longer at an intensity equating to greater than 90% maximal aerobic capacity following a 6-day supplementation protocol with ≈6 to 8 mg·kg body mass⁻¹·day⁻¹ of ${\text{NO}}_3^{-}$ via beetroot juice.

FIGURE 2.

A theoretical response of an individual cycling at a fixed workload of 200 W with and without dietary ${\text{NO}}_3^{-}$. In the absence of preexercise ${\text{NO}}_3^{-}$ supplementation, the individual is cycling at an intensity corresponding to a higher percentage of their maximal aerobic capacity (V̇O_2max). After ${\text{NO}}_3^{-}$ ingestion, the individual performs the same task, though at a lower percentage of V̇O_2max.

Although the ingestion of dietary ${\text{NO}}_3^{-}$ seems to elicit favorable effects during high-intensity aerobic exercise, it is less clear whether it improves performance during low- to moderate-intensity exercise. Laboratory-based time trial testing is one of the most reliable measures of submaximal endurance performance.⁴⁵ This type of performance-oriented task requires participants to complete a fixed distance as rapidly as possible. In this type of assessment, participants are generally blinded to factors related to their performance, including power, pace, and/or time. Dietary ${\text{NO}}_3^{-}$ has been found to elicit small, but at least marginally significant, improvements in performance of ≈0.5% to 3% for time trial tasks lasting ≈5 to 30 minutes with supplementation of ≈3 to 10 mg·kg body mass⁻¹·day⁻¹ in men and women of varying fitness levels.^15–21,46 Alternatively, no differences in performance have been detected in response to relatively higher dietary ${\text{NO}}_3^{-}$ doses in the ≈18 to 25 mg·kg body mass⁻¹·day⁻¹ range^47,48 or with longer duration time trials lasting ≈30 to 140 minutes in men and women.^20,49–56 Figure 3 provides calculated effect sizes for doses of dietary ${\text{NO}}_3^{-}$ relative to body mass resulting in endurance performance enhancements among placebo-controlled and double-blinded laboratory-based tests. From this figure, it seems the relationship between the dose of dietary ${\text{NO}}_3^{-}$ ingested and improvements in endurance performance resemble an “inverted U” function, wherein doses equivalent to approximately 6.8 to 6.9 mg·kg body mass⁻¹ may elicit the most positive effects for exercise test–related outcomes.^15,21 Thus, factors related to the amount of ${\text{NO}}_3^{-}$ consumed and/or the intensity and therefore the duration of the event may influence performance enhancement with dietary ${\text{NO}}_3^{-}$.

FIGURE 3.

Effect sizes for differences in endurance exercise performance associated with the dose of dietary ${\text{NO}}_3^{-}$ ingested within a 24-hour window before exercise.^{15–17,19–21,46} Subjects included were between the ages of 18 and 44 years and were either untrained men (□),⁴⁶ recreationally active men and women (○),²¹ recreationally active men (■),⁴⁶ and endurance trained (cyclists, runners, or triathletes) men (●).^{15–17,19,20} Effect sizes were taken from the corresponding literature, where provided. Otherwise, a Hedges g_av effect size statistic was calculated.

In practice, the intensity at which a performance trial can be maintained is inversely related to its duration. For example, endurance trained men in the study by Shannon et al²⁰ completed a 1500 m (≈5 minutes) and 10 000 m (≈45 minutes) running time trial at an intensity equivalent to ≈86% and ≈78% of their maximal aerobic capacity, respectively. After the acute ingestion of beetroot juice, performance improvement was limited to just the 1500 m time trial.²⁰ Indeed, it is possible that shorter-duration endurance events may benefit more from dietary ${\text{NO}}_3^{-}$ supplementation compared with longer-duration events. Consistent with the nature of shorter-duration events, higher-intensity physical exertion results in relatively greater cellular perturbations that decrease oxygen tension and pH in the skeletal muscle. Accordingly, short-duration (<30 minutes) endurance events may augment the reduction of ${\text{NO}}_2^{-}$ to NO after dietary ${\text{NO}}_3^{-}$ supplementation.⁵

Dietary ${\text{NO}}_3^{-}$ Supplementation and Power-Dependent Performance

Dietary ${\text{NO}}_3^{-}$ supplementation may have beneficial effects on explosive, power-dependent pursuits like sprinting²² or weightlifting.²⁹ Research again from high-quality, placebo-controlled, and double-blind ${\text{NO}}_3^{-}$ supplementation interventions indicates maximal power during single-leg knee extension, cycling, and running exercise improves with dietary ${\text{NO}}_3^{-}$ supplementation administered either acutely within 2 to 3 hours of exercise^23–27,57 or chronically for 5 to 6 days in men and women with varied training histories.^22,28 Although the exact mechanisms accounting for gains in maximal power production are largely unknown, alterations in calcium availability and/or sensitivity in the contracting muscle fibers may be responsible.⁵⁸ Dietary ${\text{NO}}_3^{-}$ doses ranging anywhere from ≈5.5 to 23 mg·kg body mass⁻¹ have been reported to favorably enhance muscle contractile function.^24,59

Such benefits related to increased maximal power with dietary ${\text{NO}}_3^{-}$ may translate to increased performance during team sport-specific tests. Whether acute or chronic, dietary ${\text{NO}}_3^{-}$ supplementation has been shown to increase the total amount of work performed^{22,29–32,60} and average power maintained³³ during intermittent high-intensity laboratory-based tests with doses equivalent to ≈4.5 to 10.4 mg·kg body mass⁻¹. However, others have shown no improvement in team sport performance tests with nitrate doses of 10.4 mg·kg body mass⁻¹ in young male basketball athletes or 10.8 mg·kg body mass⁻¹ in elite female water polo players.⁶¹ After 7 days of beetroot juice supplementation to achieve 10.4 mg of ${\text{NO}}_3^{-}\cdot \text{kg}$ body mass⁻¹·day⁻¹, Thompson et al³¹ reported a greater amount of total work performed during simulated team-sport matches as well as faster reaction times in male team-sport players. It is possible that dietary ${\text{NO}}_3^{-}$ ingestion may be more beneficial to certain types of intermittent exercise performance, as Wylie et al³³ showed a 5% increase in mean power output across repeated cycling sprints (6 seconds each) after acute ingestion of beetroot juice in male team-sport players. In contrast, no difference in mean power was observed during repeated 30- or 60-second sprints.³³ However, Dominguez et al²⁴ found that beetroot juice increased peak power, although the advantage was limited to just the first 5 seconds of a 30-second maximal cycling test in men with a history of power-dependent sports training, whereas no differences were detected during the later stages of the exercise bout.

Possible differences in muscle recruitment patterns may explain the disparities in exercise performance with dietary ${\text{NO}}_3^{-}$. Specifically, it has been suggested that dietary ${\text{NO}}_3^{-}$ may preferentially target fast-twitch skeletal muscle fibers.⁶² If so, greater performance gains during shorter-duration, explosive-type movements that require fast-twitch muscle fibers would be expected after dietary ${\text{NO}}_3^{-}$ supplementation. However, Coggan et al²⁵ found no relationship between knee extensor muscle contractile properties, that is, maximal shortening velocity or fatigue resistance (surrogate markers of muscle fiber type distribution), at baseline and the magnitude of the increase in muscle power after ${\text{NO}}_3^{-}$ intake in healthy untrained men and women. Supporting these findings, López-Samanes et al⁶³ found that the administration of a beetroot juice supplement containing 10.4 mg of ${\text{NO}}_3^{-}\cdot \text{kg}$ body mass⁻¹·day⁻¹ had no effect on the performance of movements requiring the recruitment of fast-twitch muscle fibers (ie, countermovement jump height, 10 m/20 m sprint time, agility) in young male basketball players.

Possible Factors Affecting Performance With Beetroot Juice

Endurance Training History

Regular aerobic exercise training increases the expression and activity of enzymes responsible for the endogenous synthesis of NO.^5,64 As such, individuals with a history of endurance training tend to have greater reserves of plasma ${\text{NO}}_3^{-}$ and ${\text{NO}}_2^{-}$ and NO release in response to stressors similar to those that occur during the onset of exercise.⁶⁵ Perhaps because of a higher potential for generating endogenous NO with exercise, endurance-trained athletes are not “NO limited” and consequently have less to gain from dietary ${\text{NO}}_3^{-}$ supplementation.⁴⁶ In support, Carriker et al⁴² observed reductions in oxygen consumption during submaximal exercise in low but not high aerobically fit male runners following 4 days of supplementation with ${\text{NO}}_3^{-}$. Yet, others have shown improvements in time trial performance for endurance trained and competitive endurance athletes (ie, cyclists, triathletes) after dietary ${\text{NO}}_3^{-}$ consumption.^16,17,19,20

Potential Sex Differences

Due in part to the influence of sex hormones on NO bio-availability, limited evidence suggests that women may be more likely to benefit from dietary ${\text{NO}}_3^{-}$ supplementation compared with men.²⁵ Previous work indicates that women have lower plasma ${\text{NO}}_3^{-}$ compared with men.⁶⁶ In addition, there may be differences in NO-dependent vascular function between menstrual cycle phases. When progesterone peaks during the early to mid-luteal phase of the menstrual cycle (20 ± 3 days after onset of menses), NO-dependent vascular function is reduced relative to the follicular phase (12 ± 3 days after onset of menses) when estrogen is elevated.⁶⁷ Given these differences, dietary ${\text{NO}}_3^{-}$ supplementation may increase performance-related outcomes more so in women when compared with men. In addition, a greater performance benefit with ${\text{NO}}_3^{-}$ supplementation may occur during the mid-luteal phase of the menstrual cycle. Unfortunately, the influence of sex on differences in the exercise response to dietary ${\text{NO}}_3^{-}$ supplementation remains inconclusive given that women have been largely underrepresented in this domain of research.

${\text{NO}}_3^{-}$ Reducing Agent Bioactivity

As previously mentioned, the ergogenic effects of dietary ${\text{NO}}_3^{-}$ ingestion are predominantly dependent on the reduction of ${\text{NO}}_2^{-}$ to NO. However, not all individuals respond uniformly to the ingestion of dietary ${\text{NO}}_3^{-}$.^18,49 Coggan et al²⁵ have previously reported that some individuals exhibit a profound increase (≈400%) in plasma ${\text{NO}}_2^{-}$ after the ingestion a uniform dose of dietary ${\text{NO}}_3^{-}$, whereas others failed to show any increase in plasma ${\text{NO}}_2^{-}$. It is important to note that the rise in plasma ${\text{NO}}_2^{-}$ after acute beetroot juice supplementation positively correlates with performance-related improvements.^25,49 As such, individual differences in oral microbiota and/or other reducing agents involved in the ${\text{NO}}_3^{-}-{\text{NO}}_2^{-}-\text{NO}$ pathway (eg, ${\text{NO}}_3^{-}$ reductase, stomach pH) may contribute to the between-person difference in response to dietary ${\text{NO}}_3^{-}$ supplementation.⁶⁸ Although genetic determinants may be relatively fixed, strong evidence indicates that habitual use of mouthwash may limit the effects of dietary ${\text{NO}}_3^{-}$ by destroying the anaerobic bacteria that initiate the reduction of ${\text{NO}}_3^{-}$ to ${\text{NO}}_2^{-}$ in the oral cavity.⁶⁹ Based on such data, limiting the use of mouthwash may enhance the likelihood of dietary ${\text{NO}}_3^{-}$ supplementation offering a positive effect on performance-related outcomes.

CONCLUSION

In summary, concentrated forms of dietary ${\text{NO}}_3^{-}$ particularly in the form of beetroot juice appear to elicit small but nevertheless significant improvements in the performance of endurance, high-power explosive, and high-intensity intermittent exercise. Sufficient evidence from placebo-controlled double-blinded studies suggests that dietary ${\text{NO}}_3^{-}$ may specifically benefit short-duration (ie, <30 minutes) endurance events or power-dependent, explosive movements. Doses of approximately 6 to 7 mg ${\text{NO}}_3^{-}\cdot \text{kg}$ body mass⁻¹·d⁻¹ administered either acutely within 2 to 3 hours of exercise or chronically over several days to weeks may increase the likelihood of enhanced performance. As a recommendation, additional studies should be performed to determine whether the exercise performance of certain populations (ie, women, endurance trained) benefits more from dietary ${\text{NO}}_3^{-}$ supplementation. Further ${\text{NO}}_3^{-}$ intervention studies are warranted that either control subject diet or consider the additional intake of foods containing moderate to high amounts of ${\text{NO}}_3^{-}$ using dietary recall analysis.