One of the main factors that control vasoreactivity and angiogenesis is nitric oxide produced by endothelial nitric oxide synthase (eNOS). We recently showed that knocking out eNOS induces an important reduction of mitochondrial oxidative capacity in slow-twitch skeletal muscle. Here we investigated eNOS's role in physical activity and contribution to adaptation of muscle energy metabolism to exercise conditions. Physical capacity of mice null for the eNOS isoform (eNOS−/−) was estimated for 8 wk with a voluntary wheel-running protocol. In parallel, we studied energy metabolism enzyme profiles and their response to voluntary exercise in cardiac and slow-twitch soleus (Sol) and fast-twitch gastrocnemius (Gast) skeletal muscles. Weekly averaged running distance was two times lower for eNOS−/− (4.09 ± 0.42 km/day) than for wild-type (WT; 7.74 ± 0.42 km/day; P < 0.01) mice. Average maximal speed of running was also lower in eNOS−/− (17.2 ± 1.4 m/min) than WT (21.2 ± 0.9 m/min; P < 0.01) mice. Voluntary exercise influenced adaptation to exercise specifically in Sol muscle. Physical activity significantly increased Sol weight by 22% (P < 0.05) in WT but not eNOS−/− mice. WT Sol muscle did not change its metabolic profile in response to exercise, in contrast to eNOS−/− muscle, in which physical activity decreased cytochrome-c oxidase (COX; −36%; P < 0.05), citrate synthase (−37%; P < 0.06), and creatine kinase (−24%, P < 0.01) activities. Voluntary exercise did not change energy enzyme profile in heart (except for 39% increase in COX activity in WT) or Gast muscle. These results suggest that eNOS is necessary for maintaining a suitable physical capacity and that when eNOS is downregulated, even moderate exercise could worsen energy metabolism specifically in oxidative skeletal muscle.