Nitrate is a progenitor of nitric oxide found in high concentrations in diets rich in fruits and vegetables, such as the Mediterranean. Following enterosalivary circulation, nitrate is converted by salivary bacteria to nitrite and then nitric oxide. Nitrite is a bioactive metabolite of nitric oxide hypothesised to play a role in several metabolic pathways. We explored the effect of nitrate and nitrite in skeletal muscle metabolism and mitochondrial function in mice and humans. Our aims were to assess whether the mouse model is appropriate for use to ascertain the effects of nitrate supplementation, to determine whether these effects are nitrate or nitrite dependent and to investigate whether 7 days nitrate supplementation with nitrate in humans affects metabolism and mitochondrial function. Nitrate supplementation in the mouse, increased plasma nitrate concentration but did not elicit any changes in skeletal muscle metabolism or mitochondrial function at rest. Nitrite supplementation increased plasma nitrate and nitrite concentrations and achieved an increase in metabolic efficiency manifested as a reduction in complex I-linked leak respiration at rest. Nitrate supplementation in humans for 7 days increased plasma nitrate but not nitrite concentration. Skeletal muscle metabolism and mitochondrial function after nitrate did not differ from the placebo. However, blood perfusion of muscles consisting predominantly of type I fibres was increased. Our work suggested that nitrate metabolism is similar between mice and humans and the mouse could be a good model to investigate the effects of nitrate supplementation on skeletal muscle metabolism. This work also suggests that increases in plasma nitrite concentration may be responsible for the changes observed in mitochondrial efficiency. Finally, nitrate supplementation in humans did not elicit any metabolic changes. Deliberate nitrate supplementation in order to increase performance requires further research and any recommendations on increased consumption of nitrates for this purpose may not be accurate.