Significance: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. Recent Advances: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. Critical Issues: The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. Future Directions: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker. Antioxid. Redox Signal. 23, 1144–1170.

Pharmacological sodium nitrate supplementation has been reported to reduce the O₂cost of submaximal exercise in humans. In this study, we hypothesized that dietary supplementation with inorganic nitrate in the form of beetroot juice (BR) would reduce the O₂cost of submaximal exercise and enhance the tolerance to high-intensity exercise. In a double-blind, placebo (PL)-controlled, crossover study, eight men (aged 19–38 yr) consumed 500 ml/day of either BR (containing 11.2 ± 0.6 mM of nitrate) or blackcurrant cordial (as a PL, with negligible nitrate content) for 6 consecutive days and completed a series of “step” moderate-intensity and severe-intensity exercise tests on the last 3 days. On days 4–6, plasma nitrite concentration was significantly greater following dietary nitrate supplementation compared with PL (BR: 273 ± 44 vs. PL: 140 ± 50 nM; P < 0.05), and systolic blood pressure was significantly reduced (BR: 124 ± 2 vs. PL: 132 ± 5 mmHg; P < 0.01). During moderate exercise, nitrate supplementation reduced muscle fractional O₂extraction (as estimated using near-infrared spectroscopy). The gain of the increase in pulmonary O₂uptake following the onset of moderate exercise was reduced by 19% in the BR condition (BR: 8.6 ± 0.7 vs. PL: 10.8 ± 1.6 ml·min⁻¹·W⁻¹; P < 0.05). During severe exercise, the O₂uptake slow component was reduced (BR: 0.57 ± 0.20 vs. PL: 0.74 ± 0.24 l/min; P < 0.05), and the time-to-exhaustion was extended (BR: 675 ± 203 vs. PL: 583 ± 145 s; P < 0.05). The reduced O₂cost of exercise following increased dietary nitrate intake has important implications for our understanding of the factors that regulate mitochondrial respiration and muscle contractile energetics in humans.

The purpose of this study was to elucidate the mechanistic bases for the reported reduction in the O₂cost of exercise following short-term dietary nitrate (NO₃⁻) supplementation. In a randomized, double-blind, crossover study, seven men (aged 19–38 yr) consumed 500 ml/day of either nitrate-rich beetroot juice (BR, 5.1 mmol of NO₃⁻/day) or placebo (PL, with negligible nitrate content) for 6 consecutive days, and completed a series of low-intensity and high-intensity “step” exercise tests on the last 3 days for the determination of the muscle metabolic (using³¹P-MRS) and pulmonary oxygen uptake (V̇o₂) responses to exercise. On days 4–6, BR resulted in a significant increase in plasma [nitrite] (mean ± SE, PL 231 ± 76 vs. BR 547 ± 55 nM; P < 0.05). During low-intensity exercise, BR attenuated the reduction in muscle phosphocreatine concentration ([PCr]; PL 8.1 ± 1.2 vs. BR 5.2 ± 0.8 mM; P < 0.05) and the increase in V̇o₂(PL 484 ± 41 vs. BR 362 ± 30 ml/min; P < 0.05). During high-intensity exercise, BR reduced the amplitudes of the [PCr] (PL 3.9 ± 1.1 vs. BR 1.6 ± 0.7 mM; P < 0.05) and V̇o₂(PL 209 ± 30 vs. BR 100 ± 26 ml/min; P < 0.05) slow components and improved time to exhaustion (PL 586 ± 80 vs. BR 734 ± 109 s; P < 0.01). The total ATP turnover rate was estimated to be less for both low-intensity (PL 296 ± 58 vs. BR 192 ± 38 μM/s; P < 0.05) and high-intensity (PL 607 ± 65 vs. BR 436 ± 43 μM/s; P < 0.05) exercise. Thus the reduced O₂cost of exercise following dietary NO₃⁻supplementation appears to be due to a reduced ATP cost of muscle force production. The reduced muscle metabolic perturbation with NO₃⁻supplementation allowed high-intensity exercise to be tolerated for a greater period of time.

Dietary nitrate (NO₃⁻) supplementation with beetroot juice (BR) over 4–6 days has been shown to reduce the O₂cost of submaximal exercise and to improve exercise tolerance. However, it is not known whether shorter (or longer) periods of supplementation have similar (or greater) effects. We therefore investigated the effects of acute and chronic NO₃⁻supplementation on resting blood pressure (BP) and the physiological responses to moderate-intensity exercise and ramp incremental cycle exercise in eight healthy subjects. Following baseline tests, the subjects were assigned in a balanced crossover design to receive BR (0.5 l/day; 5.2 mmol of NO₃⁻/day) and placebo (PL; 0.5 l/day low-calorie juice cordial) treatments. The exercise protocol (two moderate-intensity step tests followed by a ramp test) was repeated 2.5 h following first ingestion (0.5 liter) and after 5 and 15 days of BR and PL. Plasma nitrite concentration (baseline: 454 ± 81 nM) was significantly elevated (+39% at 2.5 h postingestion; +25% at 5 days; +46% at 15 days; P < 0.05) and systolic and diastolic BP (baseline: 127 ± 6 and 72 ± 5 mmHg, respectively) were reduced by ∼4% throughout the BR supplementation period ( P < 0.05). Compared with PL, the steady-state V̇o₂during moderate exercise was reduced by ∼4% after 2.5 h and remained similarly reduced after 5 and 15 days of BR ( P < 0.05). The ramp test peak power and the work rate at the gas exchange threshold (baseline: 322 ± 67 W and 89 ± 15 W, respectively) were elevated after 15 days of BR (331 ± 68 W and 105 ± 28 W; P < 0.05) but not PL (323 ± 68 W and 84 ± 18 W). These results indicate that dietary NO₃⁻supplementation acutely reduces BP and the O₂cost of submaximal exercise and that these effects are maintained for at least 15 days if supplementation is continued.

Dietary supplementation with beetroot juice (BR), containing approximately 5–8 mmol inorganic nitrate (NO₃⁻), increases plasma nitrite concentration ([NO₂⁻]), reduces blood pressure, and may positively influence the physiological responses to exercise. However, the dose-response relationship between the volume of BR ingested and the physiological effects invoked has not been investigated. In a balanced crossover design, 10 healthy men ingested 70, 140, or 280 ml concentrated BR (containing 4.2, 8.4, and 16.8 mmol NO₃⁻, respectively) or no supplement to establish the effects of BR on resting plasma [NO₃⁻] and [NO₂⁻] over 24 h. Subsequently, on six separate occasions, 10 subjects completed moderate-intensity and severe-intensity cycle exercise tests, 2.5 h postingestion of 70, 140, and 280 ml BR or NO₃⁻-depleted BR as placebo (PL). Following acute BR ingestion, plasma [NO₂⁻] increased in a dose-dependent manner, with the peak changes occurring at approximately 2–3 h. Compared with PL, 70 ml BR did not alter the physiological responses to exercise. However, 140 and 280 ml BR reduced the steady-state oxygen (O₂) uptake during moderate-intensity exercise by 1.7% ( P = 0.06) and 3.0% ( P < 0.05), whereas time-to-task failure was extended by 14% and 12% (both P < 0.05), respectively, compared with PL. The results indicate that whereas plasma [NO₂⁻] and the O₂ cost of moderate-intensity exercise are altered dose dependently with NO₃⁻-rich BR, there is no additional improvement in exercise tolerance after ingesting BR containing 16.8 compared with 8.4 mmol NO₃⁻. These findings have important implications for the use of BR to enhance cardiovascular health and exercise performance in young adults.

Dietary supplementation with beetroot juice (BR) has been shown to reduce resting blood pressure and the O₂cost of submaximal exercise and to increase tolerance to high-intensity cycling. We tested the hypothesis that the physiological effects of BR were consequent to its high NO₃⁻content per se, and not the presence of other potentially bioactive compounds. We investigated changes in blood pressure, mitochondrial oxidative capacity (Q_max), and physiological responses to walking and moderate- and severe-intensity running following dietary supplementation with BR and NO₃⁻-depleted BR [placebo (PL)]. After control (nonsupplemented) tests, nine healthy, physically active male subjects were assigned in a randomized, double-blind, crossover design to receive BR (0.5 l/day, containing ∼6.2 mmol of NO₃⁻) and PL (0.5 l/day, containing ∼0.003 mmol of NO₃⁻) for 6 days. Subjects completed treadmill exercise tests on days 4 and 5 and knee-extension exercise tests for estimation of Q_max(using³¹P-magnetic resonance spectroscopy) on day 6 of the supplementation periods. Relative to PL, BR elevated plasma NO₂⁻concentration (183 ± 119 vs. 373 ± 211 nM, P < 0.05) and reduced systolic blood pressure (129 ± 9 vs. 124 ± 10 mmHg, P < 0.01). Q_maxwas not different between PL and BR (0.93 ± 0.05 and 1.05 ± 0.22 mM/s, respectively). The O₂cost of walking (0.87 ± 0.12 and 0.70 ± 0.10 l/min in PL and BR, respectively, P < 0.01), moderate-intensity running (2.26 ± 0.27 and 2.10 ± 0.28 l/min in PL and BR, respectively, P < 0.01), and severe-intensity running (end-exercise O₂uptake = 3.77 ± 0.57 and 3.50 ± 0.62 l/min in PL and BL, respectively, P < 0.01) was reduced by BR, and time to exhaustion during severe-intensity running was increased by 15% (7.6 ± 1.5 and 8.7 ± 1.8 min in PL and BR, respectively, P < 0.01). In contrast, relative to control, PL supplementation did not alter plasma NO₂⁻concentration, blood pressure, or the physiological responses to exercise. These results indicate that the positive effects of 6 days of BR supplementation on the physiological responses to exercise can be ascribed to the high NO₃⁻content per se.