Endothelium-derived nitric oxide (NO) is pivotal in regulating mitochondrial O₂ consumption (V̇o₂) and glucose uptake in mice. The aim of this study was to investigate the mechanism of age- and genotype-related exercise limitation in male endothelial NO synthase (eNOS)-knockout (KO, n = 16) and wild-type (WT, n = 19) mice. Treadmill testing was performed at 12, 14, 16, 18, and 21 mo of age. V̇o₂, CO₂ production, respiratory exchange ratio, and maximal running distance were determined during treadmill running. There were good linear correlations for increase of speed with increase of V̇o₂. The difference between KO and WT mice was not significant at 12 mo but was significant at 18 mo. Linear regression showed that KO mice consumed more O₂ at the same absolute and relative workloads, suggesting that V̇o₂ was not inhibited by NO in KO mice. KO mice performed 30–50% less work than WT mice at each age (work = vertical distance × weight). In contrast to WT mice, the work performed by KO mice significantly decreased from 17 ± 1.4 m·kg at 12 mo to 9.4 ± 1.7 m·kg at 21 mo. Running distance was significantly decreased from 334 ± 27 m at 12 mo to 178 ± 38 m at 21 mo, and maximal V̇o₂, CO₂ production, and respiratory exchange ratio per work unit were significantly higher in KO than in WT mice. Gene arrays showed evidence of a fetal phenotype in KO mice at 21 mo. In conclusion, age- and genotype-related exercise limitations in maximal work performed and maximal running distance in male eNOS-KO mice indicated that fetal phenotype and age were related to onset of heart failure.