Maximal exercise-associated oxidative capacity is strongly correlated with health and longevity in humans. Rats selectively bred for high running capacity (HCR) have improved metabolic health and are longer-lived than their low-capacity counterparts (LCR). Using metabolomic and proteomic profiling, we show that HCR efficiently oxidize fatty acids (FAs) and branched-chain amino acids (BCAAs), sparing glycogen and reducing accumulation of short- and medium-chain acylcarnitines. HCR mitochondria have reduced acetylation of mitochondrial proteins within oxidative pathways at rest, and there is rapid protein deacetylation with exercise, which is greater in HCR than LCR. Fluxomic analysis of valine degradation with exercise demonstrates a functional role of differential protein acetylation in HCR and LCR. Our data suggest that efficient FA and BCAA utilization contribute to high intrinsic exercise capacity and the health and longevity benefits associated with enhanced fitness. Display omitted •High-capacity running rats (HCR) have greater fatty acid oxidation during exercise•HCR show lower mitochondrial protein acetylation at rest and with exercise•HCR have delayed accumulation of metabolic intermediates during exercise•Mitochondrial efficiency may underlie health benefits and longevity of higher VO2max Rats with high intrinsic running capacity (HCR) have improved metabolism and longevity. Overmyer et al. show that HCR efficiently use fatty acids and branched-chain amino acids during exercise and have delayed accumulation of metabolic intermediates in muscle. HCR’s enhanced mitochondrial protein deacetylation with exercise appears to augment this efficient fuel use.