Nitrate Ingestion: A Review of the Health and Physical Performance Effects

Daily lifestyle behaviors such as dietary intake, physical activity, and sleep patterns are perhaps the strongest influencing factors related to human health and athletic performance. Hence, much of the ongoing research is focused on the impact of specific lifestyle factors, including the selective use of isolated nutrients and nutritional supplements to enhance human health and functional capacity. Indeed, health enthusiasts and athletes alike are constantly searching for key nutritional ingredients and associated “super foods” that may enhance the structure and function of the body, maintain health, thwart the onset of chronic disease, and increase longevity. Indicative of increased consumer interest, the Nutrition Business Journal (NBJ) noted (from 1999 to 2009) that the US market for organic and natural foods experienced an 8.6% increase in annual growth [1]. The steadfast dietary supplement market continues to maintain pace, with NBJ appraisals of $32 billion in sales for 2012, up 7% from the prior year [1]. Current estimates indicate that the natural and organic juice market is also on the rise, with growth of 13% in 2012, yielding an astounding $2.4 billion in sales from fresh and canned artifacts [1]. Selective nutrition for health and performance outcomes is not only trending, but arguably the focal point for today’s consumers.

Consider the common beetroot (BR) (Beta vulgaris), known to be a rich source of antioxidants and micronutrients including potassium, betaine, sodium, magnesium, vitamin C and perhaps most notably, inorganic nitrate (NO₃⁻). The inorganic anion is found in generous amounts in green leafy vegetables, and also substantially found in a variety of vegetable and fruit juices including: beetroot juice (BRJ), V8^® (CSC Brands, LP, Camden, NJ, USA), carrot juice and pomegranate juice [2]. NO₃⁻ has received considerable attention in recent years and is quickly gaining traction as a health and performance enhancing nutritional supplement. NO₃⁻-rich BRJ has also recently gained popularity for proposed anti-cancer and anti-inflammatory properties, and a reduced risk for adverse cardiovascular outcomes including stroke, myocardial infarction, systemic and pulmonary hypertension, as well as the formation of gastric ulcers (Detopoulou et al. [3], Lundberg et al. 2008 [4]).

Once ingested, inorganic NO₃⁻ metabolizes in vivo to bioactive nitrite (NO₂⁻) and is subsequently salvaged and circulated in human blood [4]. Interestingly, bacteria located in the oral cavity are integral in bioactivating and reducing NO₃⁻ to NO₂⁻, through an enzymatic reduction process now commonly referred to as the enterosalivary pathway [4,5,6]. Indeed, NO₂⁻ anion is now widely recognized as a transient species that critically functions in the physiological regulation of blood flow and blood pressure [7,8]. NO₂⁻ exerts these effects in the body via its conversion to functional nitrogen oxides (NO_x), including nitric oxide (NO) [4]. NO is a diffusible free radical molecule with multiple biological functions—one of which is to signal smooth muscle within the inner lining (endothelium) of blood vessels to relax, subsequently enhancing blood flow.

Hence, the theory proposed by many companies who market “NO-stimulating supplements” is that by elevating NO, the flow of oxygenated blood to working skeletal muscle tissue may be enhanced, ultimately aiding exercise performance and recovery. To this effect, several molecules (e.g., l-arginine, carnitine, 2-nitrooxy ethyl 2-amino 3-methylbutanoate, arginine-alpha-ketoglutarate) have been marketed to the bodybuilding community as NO boosters, with proposed health and performance enhancing benefits. However, when administered orally, these molecules have only demonstrated modest evidence for elevating NO levels, with either little or no evidence of a performance improvement following supplementation. To the contrary, mounting evidence now exists surrounding the benefits of NO₃⁻ supplementation on exercise performance outcomes.

Hoon and associates recently examined the effect of NO₃⁻ supplementation on exercise performance in humans [9]. The groups’ meta-analysis examined 17 studies that utilized NO₃⁻, of which BRJ and sodium nitrate (NaNO₃⁻) were most commonly supplemented. Supplemental doses generally ranged from 300 to 600 mg and were administered as either a single bolus or up to 15 days of consistent consumption. Upon interpretation, the group showed a noteworthy modest benefit (ES = 0.79, 95% CI: 0.23–1.35) of NO₃⁻ administration on exercise performance for time to exhaustion tests (p = 0.006). The analysis also demonstrated a minor yet statistically insignificant favorable outcome on performance for time trials (ES = 0.11, 95% CI: −0.16–0.37) and graded exercise performance tests (ES = 0.26, 95% CI: −0.10–0.62). The groups’ analysis also proposed that the benefits to athletic performance were mostly seen in individuals who were inactive to recreationally active for constant load time to exhaustion performances [9]. In spite of these findings, the minor beneficial effect may be significant in an elite competitive sport setting [10], although this remains unresolved.

It has recently been reported that dietary NO₃⁻, administered in the form of BRJ or NaNO₃⁻, enhances exercise tolerance, reduces blood pressure (BP), and lowers the oxygen (O₂) cost of cardiovascular exercise [11,12,13,14,15]. Several studies indicate that NO₃⁻, through conversion to NO, is the active component within BR and other dark green vegetables responsible for regulation of BP [16,17,18,19,20,21,22,23], blood flow [16,24,25,26,27], gastric integrity [4,16,21], and the extent of tissue protection following ischemic insult [16,18,26]. The NO₃⁻ can be delivered either as a component of food—typically about 500 mL or 2 cups of BRJ [9,11,12,19,28,29,30,31,32,33,34,35,36,37], whole BR [38] or as NaNO₃⁻—typically about 10 mg/kg of body mass [15,39,40,41]. Dietary NO₃⁻ supplementation has also been shown to reduce the ATP/PCr cost associated with force production, thereby enhancing muscle contractile efficiency [11], in addition to improving the efficiency of oxidative phosphorylation [14].

Historically, NO was thought to solely be produced endogenously via the oxidation of l-arginine by nitric oxide synthases (NOS). NO₃⁻ and NO₂⁻ were believed to be mere inert metabolic end-products of this “linear” l-arginine/NO pathway. However, it is now widely accepted that food and supplement sources of NO₃⁻ act as exogenous donors of NO₂⁻, NO, and other NO_x. The recognition of this reversible step-wise reduction mechanism (NO₃⁻→NO₂⁻→NO→NO_x) is profound, predominately due to nitrite’s capacity to be reduced in blood and tissue under certain physiological conditions [7,8,24,42,43,44,45]. Specifically, conditions of low O₂ tension (e.g., ischemic and hypoxic tissue) and pH [46] in various body regions markedly promote reduction of NO₂⁻ to NO. These human physiological conditions are characterized by a largely deoxygenated venous circulation, and effectively maximize nitrite’s potency as a vasodilator in the systemic circulation, thereby producing NO in body regions of most need. Because these conditions may persist in active, exercising skeletal muscle, diet manipulation via NO₃⁻-rich sources may also provide an effective means to enhance blood flow during exercise. Hypoxic vasodilation is of particular interest to investigators and practitioners as it is a fundamental physiological process that links the flow of blood and oxygen to its demand in body tissues—a process that could potentially be utilized in both athletic and diseased populations.

In 2011, Lansley and colleagues demonstrated that 6 day supplementation (500 mL/day, ~6.2 mmol of NO₃⁻) of NO₃⁻-rich BRJ in humans reduced resting systolic blood pressure (SBP) and pulmonary O₂ consumption (V̇O₂) during walking and running exercise [13]. The authors used ion exchange resins to selectively remove NO₃⁻ from the BRJ (500 mL/day, ~0.003 mmol of NO₃⁻). In doing so, they created a placebo juice, free of NO₃⁻, yet identical in terms of color, taste, smell, and texture. This allowed for a targeted analysis of NO₃⁻ in regards to its effect on blood pressure and the physiological responses to exercise. The selective removal of NO₃⁻ from BRJ resulted in no changes to plasma NO₂⁻ concentration, blood pressure, or the physiological responses to exercise [13]. These findings suggest that inorganic NO₃⁻ anion is likely an essential nutrient found in vegetable rich diets. Moreover, these findings indicate that the reductions to SBP and V̇O₂ are principally attributable to the high NO₃⁻ content of BRJ (>250 mg/100 g of fresh weight) and not the other constituents [13].

In some cases, supplementation has shown to improve one’s tolerance to exercise [30,37,47]. These studies effectively demonstrate a health and performance-enhancing role for dietary NO₃⁻ supplementation. Particularly, NO₃⁻ ingestion may prove viable as an adjunct therapeutic agent for treatment and prevention of hypertension, and more generally “blood-oxygen” delivery disorders commonly associated with metabolic syndrome and the onset of chronic disease [4]. As such, several nutritional supplements containing NO₃⁻ have recently been introduced to the consumer market, claiming various improvements in health and exercise performance.

Considering the increasing interest in NO₃⁻ supplementation, the purpose of this review is to provide an up to date summary of research pertaining to dietary NO₃⁻ supplementation for purposes of enhancing human health and performance. This review includes studies involving both normal environments and those of hypoxic conditions.

Initially described by Roy and Brown in 1879, hypoxic vasodilation is a preserved physiological reaction to inadequate regional or gross body O₂ supply [48]. Effectively, it is the body’s means of coupling blood flow and, hence, O₂ distributions to tissue metabolic demand. Hypoxic vasodilation is dependent upon a detection mechanism whereby specific feedback vasodilator operators identify discrepancies between tissue O₂ consumption and “real-time” oxygenated blood delivery [49]. The specific elementary reactions inherent to this O₂ sensing mechanism and many of the identities of key vasodilator effectors went mostly undetected for several years [7].

Dating back to 1953, however, Furchgott and Bhadrakhom [50] demonstrated nitrite’s ability to vasodilate pre-constricted rabbit aortic vessels. Even in the 1970s and early 1980s, Murad’s group and Ignarro’s group mirrored these earlier findings; upholding the demonstrated vasodilatory potential of NO₂⁻ in aortic rings via activation of soluble guanylyl cyclase [51,52,53]. These pioneering scientists utilized micromolar to millimolar concentrations of NO₂⁻ to achieve these vasodilatory effects in vitro. It was also shown in 1994 by two groups independently that NO is generated in the stomach from acidified NO₂⁻ [42,43]. The NO generated in the acidic environment of the stomach is around 10–100 ppm and is more than sufficient, actually several magnitudes higher than the limiting threshold necessary to induce vasodilation. However, due to the fact that its vasodilatory properties were only exhibited at concentrations several orders of magnitude higher than that seen physiologically, the interest in nitrite’s vasodilatory potential was limited.

Lauer and colleagues demonstrated in 2001 that NO₂⁻ exhibited no capacity to vasodilate when infused into human forearms at concentrations of 200 μmol/L [54]. Hence, given the nanomolar concentrations present in mammalian blood, NO₂⁻ was negated as an effective blood vessel dilator as late as the early 2000’s [54,55,56]. In spite of apparent NO₂⁻ inactivity, Gladwin and colleagues identified an arterial-venous gradient in NO₂⁻ which markedly increased consumption of NO₂⁻ during exercise, in which the NOS pathway is hindered in humans [7]. These findings led the group to propose a now broadly accepted concept—NO₂⁻ is a likely precursor of NO, preferentially reduced to NO in anoxic and acidic environments [4,46].

Modin and colleagues demonstrated that NO₂⁻ functioned to dilate aortic ring preparations at physiological concentrations when the pH in the medium mirrored ischemic and metabolically active tissues [46]. In 2003, Cosby and associates showed that infused NO₂⁻ in extremely minor micromolar concentrations significantly improved blood flow in human circulation—an effect that was pronounced when subjects underwent exercise performance [24]. Hence, the past 15 years have proved extremely monumental as it is now apparent that blood and tissue NO₂⁻ is reduced in vivo to form NO and modify blood flow [7,24].

Recent studies performed by Ferguson and colleagues [57,58] provide insight into the role of beetroot juice to improve metabolic control. For example, one study focused on skeletal muscle hindlimb (composed of 28 muscles and muscle parts) blood flow and vascular conductance in 19 adult male Sprague-Dawley rats during submaximal treadmill running [58]. At random, rats were assigned to consume either tap water or BR-infused water for a 5 day period with a NO₃⁻ dose of approximately 1 mmol/kg/day. The BR was diluted in 100 mL of tap water and the rat consumption was observed on a daily basis to ensure plasma NO₃⁻ and NO₂⁻ levels corresponded to those seen in human studies. Compared to the control group, the BR group showed greater elevations in total exercising skeletal muscle blood flow and vascular conductance. Of note, the NO₃⁻ supplemented group demonstrated enhanced blood flow and vascular conductance to primarily fast-twitch type II + d/x muscle fibers [58]. However, for the first time, despite a ~10 mmHg reduction in mean arterial pressure (MAP), changes in exercising skeletal muscle blood flow were positively augmented. These findings confirm the role of NO₃⁻ in the regulation of muscle O₂ perfusion and are likely the result of its reduction to NO₂⁻ and subsequent reduction to NO in vivo. Moreover, these findings suggest that increased blood flow and hence increased O₂ delivery may sufficiently increase the driving pressure of O₂ across the capillary-myocyte interface [58].

These improvements to blood flow and vascular conductance to skeletal muscle were observed without any decreases to the splanchnic circulation. Hence, the authors proposed that the enhanced skeletal muscle blood flow and vascular conductance were likely due to improved cardiac output and stroke volume rather than blood redistribution effects from vasoconstriction mechanisms of the splanchnic circulation. These data provide evidence that NO₃⁻ ingestion increases muscle O₂ delivery in a fiber-dependent manner post-reduction to NO₂⁻ and NO.

To this effect, it is apparent why modulating the NO₃⁻→NO₂⁻→NO via the diet may prove beneficial for physiologists, dieticians, nutritionists, and physicians alike.

Nitrate (NO₃⁻) is an inorganic polyatomic anion that exists naturally in the environment. It is present in both air and drinking water, as well as in certain foods, and is produced endogenously from a family of nitric oxide synthases (NOS) via oxidation of the amino acid l-arginine. Nitrate is found primarily in the diet (>80%) as an inorganic component of vegetables [4]. Predominate sources of nitrates are beets, celery, lettuce, radishes, and spinach [59] and are rather prevalent in green vegetables produced in heated glasshouses or hydroponically grown [60]. NO₃⁻, through conversion to nitrite (NO₂⁻), is also a common food preservative found with bacon, bologna, hot dogs, and luncheon meats [61]. Nitrite consumption is mainly found exogenously via consumption of cereal products, vegetables, and cured meats [61,62]. NO₃⁻ and NO₂⁻ have been used for centuries to maintain flavor and color in processed and cured meats [16], and have demonstrated anti-microbial [63] and anti-fungal properties [64]. In the form of potassium NO₃ (saltpeter), the anion is in fact a natural impurity of salt, and historically contributed to the pinkish-red hue observed in salted meats [16].

Following oral consumption, NO₃⁻ is quickly absorbed in the duodenum and jejunum and is dispersed amongst the whole body [65]. Post-absorption, NO₃⁻ begins circulation within plasma and has a half-life of about 5 h. Following uptake from the blood, NO₃⁻ is concentrated in saliva via an active transport system. The NO₃⁻ is concentrated by at least 10-fold in saliva and is capable of reaching concentrations up to several millimolar [66]. The explanation for why NO₃⁻ is concentrated in human saliva remains largely uncertain [16], but it is estimated that up to 25% of dietary NO₃⁻ is salvaged in this enterosalivary circulation [16]. The larger portion is excreted via the kidneys. Once secreted in the oral cavity, commensal bacterial anaerobes—located predominantly in the crypts of the tongue—bioactivate NO₃⁻ and reduce it to NO₂⁻ in saliva. Roughly 20% of this salivary NO₃⁻ is reduced, which translates to approximately 4%–8% of the ingested NO₃⁻ dose [66,67,68]. Using NO₃⁻-reductase enzymes, these oral microfloras use NO₃⁻ as an alternate one electron acceptor and reduce it to NO₂⁻ [5].

Once swallowed, salivary NO₂⁻ has demonstrated ability to convert to NO in the acidic stomach [42,43], but it is also evident that some of this NO₂⁻ is absorbed to increase circulating plasma NO₂⁻ concentrations [4]. Many tissues and enzymatic systems of the body have been described to reduce NO₂⁻; a number of hemeproteins (e.g., myoglobin, hemoglobin, xanthine oxidase) also procure capacity to reduce NO₂⁻ [69]. Additionally, it is also known that oxygenated myoglobin and hemoglobin effectively seek out NO and oxidize it to NO₃⁻. It has been estimated that NO yield rates may be as fast as one to tens of nanomolar per second under physiological conditions [69]. Additionally, it is apparent that some NO₂⁻ conversion to NO is found in the gut and is likely due to its innate diverse characteristics (e.g., large latitudes of pH and oxygen partial pressures, gastric content). For a detailed review of the mechanisms of action of nitrate metabolism, refer to [10,69].

Hyde and colleagues recently measured the NO₃⁻ reducing capacity of tongue-scraping samples from 6 healthy volunteers, and analyzed the metagenomes of the microbial communities to identify bacteria that predominately contribute to NO₃⁻ reduction in the oral cavity. The group found a number of bacterial genera that significantly contributed to reduction of NO₃ˉ including: Veillonella, Neisseria, Haemophilus, Porphyromonas, Fusobacterium, Prevotella and Leptotrichia [6]. Secondly, utilizing a whole genome shotgun sequencing approach, the group also identified 14 candidate species that significantly contributed to NO₃⁻ reduction. Interestingly, the groups’ use of a community approach allowed for specific identification of bacteria that may directly or indirectly influence NO₃⁻ bioactivation in the oral cavity. Fundamental questions still remain as to whether or not specific bacterial communities correlate to NO insufficiency and an inflated risk for cardiovascular disease. In this regard, multi-biofilm analyses and larger subject groups are needed to truly elucidate specific bacteria integral to NO₃⁻ bioactivation as this will likely reveal stronger correlations between oral health and systemic disease [6]. Additionally, it is likely that oral bacterial communities differ amongst different races, patient populations, and between individuals in different geographical locations [6]. If this is indeed so, enhancement of bacterial communities specific for NO₃⁻ reduction in patient populations may prove beneficial.

Further, the use of certain mouthwashes may be urged against if such bacterial treatments are used in the future. This is due to the fact that chlorhexadine-based mouthwashes have shown to inhibit response to NO₃⁻ and actually heighten BP values in both animal and human studies, implicating the necessity of oral bacteria for NO₃⁻ bioactivation [18,21]. It is still unclear whether alcohol-based mouthwashes and chlorinated swimming pools exert similar negative BP effects as those seen in chlorhexidine-based mouthwash studies; perhaps these may also be considered in the foreseeable future [6].

In order to determine the amount of ingested NO₂⁻ that actually reached that reaches the systemic circulation in humans, Hunault and others [70] supplemented 9 volunteers with single sodium NO₂⁻ doses equal to approximately 150 and 300 mg (0.06 mmol NaNO₂ and 0.12 mmol NaNO₂/mmol of Hb, respectively). The volunteers were given NaNO₂⁻ in aqueous form, in an effort to most closely mirror what happens physiologically, as the majority of human exposure to NO₂⁻ is a result of salivary reduction of NO₃⁻ [62]. The authors concluded that the absolute bioavailability of NO₂⁻ (i.e., the amount that reached the systemic circulation) was 95% and 98% respectively for the two doses. These findings suggested to the group that NO₂⁻ is almost completely absorbed in the duodenum and jejunum and that its first pass effect through the liver is minimal at best [70].

Before discovery in the 1980s of endogenous NO biosynthesis, it was inconceivable that cells would purposely produce a potentially toxic molecule like NO, also known as endothelium-derived relaxing factor. The colorless and odorless gas is considered a reactive nitrogen species and is a common pollutant in air. It is also a component in cigarette smoke, and a toxic gas found in the exhaust of cars and jet planes known to generate acid rain and eradicate the ozone [16]. However, the picture is now clear that NO plays numerous biological roles when available at physiological concentrations, functioning and affecting neurotransmission, wound healing, tumors, asthma, glaucoma, hemoglobin delivery of oxygen, blood flow, thrombosis (i.e., blood clotting), muscle contractility, differentiation and proliferation of stem cells, glucose and calcium homeostasis, and mitochondrial O₂ consumption, while also being associated with learning and memory formation [17,25,71]. The gas is also a principal neurotransmitter that mediates inflammation, host defense, and even penile erectile function [16]. It should be noted that this list represents a small fraction of the many biological processes that are inherent to NO. Though the molecule possesses a very simple molecular structure, the breadth of NO involvement in the human body remains a topic of continued investigation.

NO is primarily of interest to investigators and practitioners due to its potential for relaxing human vasculature [16]—an effect that may result in improved blood flow during rest [27] and exercise [27,72]. Vasodilation is paramount to human health, as the process itself is utilized by the body to deliver oxygenated blood to areas of the body that are in greatest need (e.g., contracting skeletal muscle, beating hearts, ischemic areas, etc.). Vasodilation is not solely used for delivery of oxygenated blood, but also for the delivery of glucose, lipids, and other nutrients to a variety of tissues.

Given the wide variety of biological processes known to be regulated by NO (mainly the ability of NO to augment blood flow), it is apparent why maintaining a continuous circulating pool of NO might be beneficial to both athletes and those who suffer from ischemia and hypoxia-related conditions. Because NO is a gas, produced endogenously from the amino acid l-arginine—by three isoforms of NOS, it is very difficult to provide oral supplementation of NO directly (Note: oral supplementation with l-arginine is also largely ineffective for elevating circulating NO). However, through consumption of dietary supplements and certain donor drugs, as well as through intake of vegetables rich in NO₃⁻, clinicians and exercise scientists have evaluated alternative methods to increase circulating NO, independent of its endogenous NOS biosynthesis.

As a therapeutic agent and NO donor, inorganic nitrate and nitrite act to dilate blood vessels. However, analysis of the pharmacodynamic profiles between organic and inorganic nitrate show striking differences for inducing vasodilation in blood vessels, particularly in regards to potency. Inherent to pharmaceutical agents like nitroglycerine and isosorbide mononitrate (e.g., organic nitrate) is their capacity to act as lipophilic dilators, converting rapidly to NO in smooth muscle via the mitochondrial enzyme, aldehyde dehydrogenase [69]. Inorganic nitrate and nitrite also actively convert to NO and induce vasodilation, but pharmacodynamically exert their dilatory effect at much lower orders of magnitude. Organic nitrate is frequently used in the cardiovascular field of medicine and can be administered via several forms (e.g., tablet, transdermal patch, ointment, in solution for intravenous purposes, as an aerosol spray delivered sublingually) [16]. However, it is well known in the medicinal field that nitrovasodilators such as nitroglycerin are subject to tolerance in human patients and may, over time, decrease their effectiveness for promoting vasodilation [16].

In this regard, inorganic nitrate and nitrite may prove rather advantageous as possible contenders for future treatment and prevention of ischemia-related illnesses. Additionally, the conversion from inorganic NO₂⁻ to NO occurs along a pH and pO₂ gradient (e.g., acidic and oxygen-deficient environments [46]. At such high concentrations, NO and its reactions products are toxic to many enteropathogens (e.g., Salmonella, Shigella, Helicobacter pylori) and principle mediators of host defense [73]. Many enzymes possess the capacity to reduce NO₂⁻ to NO, however none may be as important as the enzymes innate to the hemeproteins myoglobin (tissue) and deoxyhemoglobin (plasma) along the oxygen gradient. NO₂⁻ likely reacts with myoglobin and deoxyhemoglobin via a similar mechanism where its reduction to NO is enhanced in anoxic and acidic conditions [7]. NO functions to dilate arterioles and allow for more O₂-carrying-Hb to reach hypoxic and ischemic tissue beds [51,52]. The pH and pO₂ gradient is of paramount importance as NO₂⁻ has been shown to enhance blood flow selectively in anoxic environments and may allow for blood to flow to areas where it is most necessary [7,58,71].

Nitrate from dietary sources may be paramount in the future of cardiovascular medicine by allowing generation of NO to localized areas experiencing hypoxia (e.g., oxygen deficiency) and ischemia (blood deficiency). Hypothetically, inorganic nitrate may match NO to areas most needed rather than globally making NO available throughout the body and running the risk of hypotension from whole-body vasodilation. Perhaps most intriguing is that the amount of inorganic NO₃⁻ to effectively manipulate the NO₃⁻→NO₂⁻→NO pathway and promote the NO-pool can be obtained via a diet rich in vegetables [4,16].

Aside from its vasodilatory potential, NO acts to decrease myointimal hyperplasia [74,75,76]. This condition is characterized by an abnormal narrowing of arteries resulting from proliferation and migration of vascular smooth muscle cells as well as expansion of extracellular matrix in the intima of affected arteries. NO plays a key role in this pathophysiological process by inhibiting adherence and infiltration of monocytes and can even signal apoptosis of affected smooth muscle cells. Furthermore, an increase in circulating levels of NO₂⁻/NO may protect hypoxic tissues (e.g., damaged blood vessels, local ischemic areas, contracting skeletal muscle) by improving blood and O₂ perfusion to ischemic areas. The increase in blood flow would theoretically stimulate O₂ and nutrient delivery (e.g., amino acids and glucose) to exercising skeletal muscle, hence aiding exercise performance [28]. Several mechanisms have been proposed for this NO₂⁻-derived NO matching of blood flow to areas experiencing O₂ deprivation [28,55,77,78]. Due to this significant capacity for improving human circulatory system, NO-stimulating therapeutics and NO₃⁻ supplements are novel targets for cardiovascular health investigators, clinicians, and sport supplement companies alike [4,8].

As reported by the World Health Organization, cardiovascular disease accounts for 40% of all deaths. Astoundingly, in 2006, total health care costs in the US were upwards of $2 trillion dollars (~$7000 dollars/person) and expected to double by 2015 according to the National Healthcare Statistics Group. The costs alone associated with chronic “lifestyle” diseases that include obesity, diabetes, hypertension, and coronary artery disease are responsible for 75% of the nation’s annual cost of health care [16]. Many of these chronic diseases involve a malfunctioning endothelium and an inability to adequately produce and maintain NO homeostasis [16]. In this regard, a new trend has seemed to emerge in the nutrition arena, suggesting that along with reduced caloric and fat intake, diets rich in food that stimulate NO bioavailability (e.g., NO₃⁻ and NO₂⁻-enriched foods) are viable options for seeking a variety of health benefits. Indeed, along with daily physical activity, dietary intake should be considered a first-line target for disease prevention [16].

In the United States, dietary intake of NO₃⁻ has been estimated to be about 40 to 100 and 31–185 mg/day, with approximately 85% coming solely from vegetables in the diet [59,79]. This intake can be greatly enhanced to reach more than 1200 mg (~20 mmol) by including high NO₃⁻ foods, as a part of the Dietary Approaches to Stop Hypertension (DASH) diet. Interesting to note, this intake is approximately 5-fold higher than the World Health Organization’s Acceptable Daily Intake (ADI) of 3.7 mg of NO₃⁻ per kg of body weight per day and more than a 2-fold higher than the United States Environmental Protection Agency of 7.0 mg of NO₃⁻ per kg of body weight per day [2]. Some DASH studies have proposed that the diet may be as efficacious as any single hypotensive agent in regards to reducing BP [80].

Perhaps most intriguing, however, is that nearly identical (and sometimes even greater) reductions have been seen with moderate doses of dietary NO₃⁻ [20,23]—although much more research is needed to definitively state this [16]. Indeed, NO₃⁻ is likely the major component of vegetable-rich diets and likely one of many responsible for the beneficial responses to BP. Yet, until recently, the anion has remained largely ignored. However, it is apparent that it is an essential nutrient inherent to vegetable and fruit-rich diets such as Mediterranean and traditional Japanese diets, that have long been affiliated with protection against cardiovascular disease and diabetes mellitus type 2 [16].

A recent study was performed by Hoon and colleagues [100] that tested NO₃⁻ supplementation in national-level cyclists and their response to high-intensity exercise. Using two cycle ergometer tests with each lasting 4 min, 26 cyclists performed the test followed by a 75 min rest interval. Intriguingly, the authors utilized a “multi-dose” approach—in which each cyclist performed the exercise test under all 4 separate dosing regimens. The separate conditions consisted of: 70 mL of NO₃⁻-rich BRJ 150 min or 75 min before the initial cycling time trial, and an additional “booster-dose” of 35 mL in the 150 min condition post-first trial and a placebo. Although the mean findings in the 3 NO₃⁻ groups were inconclusive for the first trial, the authors found that the subsequent cycling trial was potentially harmful. They also noted a lack of consistency within individuals. These findings suggest that NO₃⁻ may be particularly ineffective and perhaps even detrimental to high-intensity cycling exercise those performing at a competitive level.

Indeed, Many authors have now reported that NO₃⁻ supplementation (mainly administered via BRJ) can improve finish times [9,29], elongate time to exhaustion [14,28,30,37], reduce the gross O₂ cost of exercise [12,28,30,31,101], and increase peak power [22,29,36] and work rate [12,13,28,36]. With consideration for the above, it should also be noted that other authors have also reported no ergogenic effect of NO₃⁻ supplementation [32,38,39,40]. Many of these studies have also involved cycling exercise performed by well-trained or elite-level athletes. These will be considered within the remaining sections of this paper, which highlight the use of NO₃⁻ supplementation to improve exercise performance (e.g., walking, running, arm and leg cycling, rowing, static apnea tests).

This review focused primarily on the impact of dietary nitrate to aid physical performance and covered 31 studies, inclusive of over 300 participants. Quantitative analysis suggests that performance enhancing benefits are noted predominately with doses ranging from 5 to 9 mmol of NO₃⁻, delivered as either a single bolus or as multiple (e.g., 1–15) daily servings of said dosage ingestion. This correlates to about 500 mL (~2 cups) of BRJ (~5–9 mmol of NO₃⁻) at each dosing, with peak elevations in plasma NO₂⁻ resulting 2.5 and 3 h post-ingestion. These peak plasma NO₂⁻ concentrations appear to directly correlate with peak reductions in systolic and diastolic blood pressure and may be related to peak physical performance as well. Benefits have been noted with up to 15 days of chronic NO₃⁻ supplementation and appear to persist 24 h post-ingestion. No additional benefits have been noted by increasing NO₃⁻ concentration above 8.4 mmol and up to 18.1 mmol of NO₃⁻, although further research is needed in this area.

Dietary intake is perhaps the most important environmental variable in controlling our overall health. It is particularly relevant to analyze foods on the basis of their constituent ingredients and specifically how these ingredients act (e.g., synergistically, additively, or negatively) within the human body. The interplay of NO₃⁻ and other nutrients commonly found in vegetables and BRJ (e.g., vitamin C, polyphenols and fatty acids) remains an important area of investigation as these interactions may amplify or repress the health and performance effects associated with NO₃⁻.

Growing evidence suggests that ingestion of inorganic nitrate—richly found in beetroot juice—enhances blood flow in human and animal microvasculature and leads to reductions in BP, both systolic and diastolic. NO₃⁻ and the BP benefits following intake also appear to be sex-dependent which may be due differences amongst in vivo handling or incident lingual bacteria. Additionally, moderate dietary NO₃⁻ has demonstrated capacity to impede platelet function, prevent endothelial dysfunction following ischemia-reperfusion and decrease the O₂ cost associated with exercise. In some cases moderate dietary NO₃⁻ also appears to enhance exercise tolerance. Indeed, NO₃⁻ mostly in the form of BRJ and NaNO₃ has shown remarkable capacity to reduce blood pressure and may provide relief to those experiencing hypertension, hypoxia, and ischemia These findings are paramount to the field of medicine as many patients suffer from blood flow disorders that impair their ability to exercise at low intensities (e.g., walking or standing for long periods). The declining functional capacity that is associated with these cardiovascular disorders greatly impairs an individual’s ability to maintain a satisfactory quality of life. In this regard, ingestion of dietary NO₃⁻, simply obtained by diets rich in vegetables or by drinking about two cups of BRJ, may provide a low-cost treatment for individuals with blood flow disorders.

As such, individuals suffering from hypertension, peripheral artery disease and ischemia-related diseases (e.g., blood flow disorders) may be relieved by regular consumption of nitrate-rich vegetables—or perhaps using beetroot juice as a source for enhancing blood flow both at rest and during exercise. Individuals seeking said benefits should note that ingestion of 500 mL (~2 cups) of BRJ provides sufficient NO₃⁻ (~5–9 mmol) content to provide ergogenic and health benefit. Athletes looking to utilize BRJ for ergogenic aid should note that BRJ should be consumed approximately 2.5–3 h prior to an exercise bout. A picture is emerging supporting increased intake of NO₃⁻-rich natural vegetables and beetroot juice as a low cost and natural method for the prevention and treatment of cardiovascular disease and ischemia-related diseases. These findings are important for cardiovascular medicine and may provide relief to those individuals with impaired blood flow; allowing them to exercise at low-intensities for longer periods of time and providing them with improved quality of life.

Of interest to athletes, health enthusiasts, and physicians alike is the NO₂⁻/NO demonstrated capacity to elicit several responses in human (and animal) vasculature and skeletal muscle that result in a: (1) reduced gross O₂ cost of cardiovascular exercise; (2) improved exercise tolerance; (3) reduced ATP cost of force production; (4) improved blood flow and nutrient delivery; and (5) improved revascularization in mice induced with chronic ischemia. This, along with other growing bodies of evidence, suggests a role for the NO₃⁻→NO₂⁻→NO pathway in hypoxic tissue (e.g., exercising muscle, ischemic areas) with regards to vasodilation and perfusion.

The available evidence indicates that NO₃⁻ supplementation for moderate performance improvement in constant-load and incremental-load time trial and time to exhaustion tasks such as walking, running, rowing, and cycling could prove beneficial—for inactive to well-trained individuals and even for patients with PAD. These benefits may prove meaningful in elite sport and ischemia-related diseased contexts. Several authors have reported dose-dependent relationships that are likely related to individual training status, number or type of incident NO₃⁻-reducing microflora, exercise mode, or supplementation period. It is also important to note that when supplementing NO₃⁻ in human subjects, it may be of value to determine if these individuals regularly use antibacterial mouthwash or swim in chlorinated pools, as both may potentially inhibit the activity of NO₃⁻-reducing bacteria, impacting the response to NO₃⁻ supplementation.

Additional research is needed to determine the impact of NO₃⁻ supplementation on anaerobic exercise performance—specifically resistance exercise of high volume—as individuals performing such exercise are often the main target market for many dietary supplements containing NO₃⁻. Studies should also seek to identify the synergistic relationship between isolated NO₃⁻ and other ingredients found in NO₃⁻-rich vegetables, to explore the specific dose-response relationships needed to elicit health and ergogenic benefits, and to prolong the supplementation period beyond a relatively short period (i.e., >15 days) to determine if more robust effects can be observed with longer-term treatment. Finally, as more individuals are becoming interested in routine use of NO₃⁻ supplementation, thoroughly examining the long-term safety profile of NO₃⁻ supplementation should be considered.

WTC was chiefly responsible for performing the literature search associated with this manuscript. WTC, SRL, and RJB contributed to the writing of the manuscript, as well as the multiple rounds of editing made to the manuscript.

The authors declare no conflict of interest.