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Dietary nitrate (NO 3 −) supplementation can increase nitric oxide (NO) bioavailability, reduce blood pressure (BP) and improve muscle contractile function in humans. Plasma nitrite concentration (plasma [NO 2 −]) is the most oft-used biomarker of NO bioavailability. However, it is unclear which of several NO biomarkers (NO 3 −, NO 2 −, S-nitrosothiols (RSNOs)) in plasma, whole blood (WB), red blood cells (RBC) and skeletal muscle correlate with the physiological effects of acute and chronic dietary NO 3 − supplementation. Using a randomized, double-blind, crossover design, 12 participants (9 males) consumed NO 3 −-rich beetroot juice (BR) (∼12.8 mmol NO 3 −) and NO 3 −-depleted placebo beetroot juice (PL) acutely and then chronically (for two weeks). Biological samples were collected, resting BP was assessed, and 10 maximal voluntary isometric contractions of the knee extensors were performed at 2.5–3.5 h following supplement ingestion on day 1 and day 14. Diastolic BP was significantly lower in BR (−2 ± 3 mmHg, P = 0.03) compared to PL following acute supplementation, while the absolute rate of torque development (RTD) was significantly greater in BR at 0–30 ms (39 ± 57 N m s−1, P = 0.03) and 0–50 ms (79 ± 99 N m s−1, P = 0.02) compared to PL following two weeks supplementation. Greater WB [RSNOs] rather than plasma [NO 2 −] was correlated with lower diastolic BP (r = −0.68, P = 0.02) in BR compared to PL following acute supplementation, while greater skeletal muscle [NO 3 −] was correlated with greater RTD at 0–30 ms (r = 0.64 , P= 0.03) in BR compared to PL following chronic supplementation. We conclude that [RSNOs] in blood, and [NO 3 −] in skeletal muscle, are relevant biomarkers of NO bioavailability which are related to the reduction of BP and the enhanced muscle contractile function following dietary NO 3 − ingestion in humans. • It is unclear which NO biomarkers in blood and muscle correlate with the physiological effects of acute and chronic dietary nitrate supplementation. • In a crossover design, participants consumed NO 3 −-rich beetroot juice (BR) and NO 3 −-depleted placebo (PL) acutely and then for two weeks. • Greater whole blood [RSNOs] was correlated with lower diastolic BP with BR, while greater skeletal muscle [NO 3 −] was correlated with greater rate of muscle torque development with BR. • RSNOs in blood, and NO 3 − in skeletal muscle, are relevant biomarkers of NO bioavailability which are related to the physiological effects of dietary NO 3 − ingestion in humans. [ABSTRACT FROM AUTHOR]