Purpose: To investigate whether chronic supplementation with a low or moderate dose of dietary nitrate (NO3-) reduces submaximal exercise oxygen uptake (VO2) and to assess whether or not this is dependent on acute NO3- administration prior to exercise. Methods: Following baseline tests, 34 healthy subjects were allocated to receive 3 mmol NO3-, 6 mmol NO3- or placebo. Two hours following the first ingestion, and after 7, 28 and 30 days of supplementation, subjects completed two moderate-intensity step exercise tests. On days 28 and 30, subjects in the NO3- groups completed the test 2 h post consumption of a NO3- dose (CHR + ACU) and a placebo dose (CHR). Results: Plasma nitrite concentration ([NO2-]) was elevated in a dose-dependent manner at 2 h, 7 days and 28-30 days on the CHR + ACU visit. Compared to pre-treatment baseline, 6 mmol NO3- reduced the steady-state VO2 during moderate-intensity exercise by 3% at 2 h (P = 0.06), 7 days and at 28-30 days (both P < 0.05) on the CHR + ACU visit, but was unaffected by 3 mmol NO3- at all measurement points. On the CHR visit in the 6 mmol group, plasma [NO2-] had returned to pre-treatment baseline, but the steady-state VO2 remained reduced. Conclusion: Up to similar to 4 weeks supplementation with 6 but not 3 mmol NO3- can reduce submaximal exercise VO2. A comparable reduction in submaximal exercise VO2 following chronic supplementation with 6 mmol NO3- can be achieved both with and without the acute ingestion of NO3- and associated elevation of plasma [NO2-]. (C) 2016 Elsevier Inc. All rights reserved.