Dose-response Effects of Dietary Nitrate on Running Time-to-exhaustion and Neuromuscular Fatigue: a Randomized Crossover Exploratory Pilot Study

Dietary nitrate (NO₃-), naturally present in vegetables such as beetroot and leafy greens, can be converted into nitric oxide (NO) through the nitrate-nitrite-NO pathway. Nitric oxide plays an important role in regulating blood flow, muscle contractility, mitochondrial efficiency, and oxygen utilization during exercise. For these reasons, dietary nitrate supplementation has attracted considerable interest as a nutritional strategy to enhance exercise performance.

Previous studies have reported improvements in endurance performance following acute dietary nitrate supplementation, particularly with doses between 5 and 9 mmol. However, the optimal dose for maximizing potential ergogenic effects remains uncertain, especially in recreationally trained individuals. Furthermore, limited evidence is available regarding the dose-response relationship between nitrate supplementation and exercise-induced neuromuscular fatigue.

This pilot study was designed to evaluate the acute effects of three different doses of dietary nitrate compared with placebo using a randomized, double-blind, placebo-controlled crossover design. Each participant completed four experimental trials, receiving placebo or one of three nitrate doses in a randomized order before performing an identical treadmill running test to exhaustion at the speed corresponding to the second ventilatory threshold (VT2). Neuromuscular performance, blood lactate concentration, and perceived exertion were assessed before and after exercise using standardized procedures.

The findings of this study are expected to contribute to a better understanding of the dose-response relationship of acute dietary nitrate supplementation and to support the design of larger clinical trials investigating its effects on endurance performance and neuromuscular function.

Inclusion criteria

• Male adults aged 18 to 40 years.
• Recreationally trained, performing endurance or combined endurance/resistance exercise at least 3 times per week for the previous 6 months.
• Maximal oxygen uptake (VO₂max) consistent with recreational training status.
• Able to perform maximal treadmill running exercise.
• Willing and able to comply with all study procedures.
• Provided written informed consent before participation

Exclusion criteria

• Current smoking or use of nicotine products.
• History of cardiovascular, pulmonary, metabolic, neurological, or musculoskeletal disorders that could affect exercise performance or participant safety.
• Current use of medications or dietary supplements known to influence nitric oxide metabolism or exercise performance (e.g., nitrate supplements, phosphodiesterase-5 inhibitors, antibacterial mouthwash).
• Allergy or intolerance to beetroot or any component of the study supplements.
• Following a vegetarian or vegan diet, or habitually consuming a high-nitrate diet.
• Consumption of alcohol, caffeine, or strenuous physical exercise within the period restricted by the study protocol before each experimental visit.
• Failure to comply with pre-test dietary or behavioral instructions. Participation in another clinical trial within the previous 30 days.
• Any condition that, in the opinion of the investigators, could interfere with study participation or interpretation of the results.