As substrate for evaluation of metabolic diseases we developed novel rat models that contrast for endurance exercise capacity. Through two-way artificial selection we created rodent phenotypes of intrinsically low- (LCR) and high-capacity runners (HCR) that also differ markedly for cardiovascular and metabolic disease risk factors. Here we determined skeletal muscle proteins with putative roles in lipid and carbohydrate metabolism to better understand the mechanisms underlying differences in whole-body substrate handling between phenotypes. Animals (generation 16) differed for endurance running capacity by 295%. LCR had higher resting plasma glucose (6.58 ±0.45 vs. 6.09 ±0.45 mmol/L), insulin (0.48 ±0.03 vs. 0.32 ±0.02 ng/mL), non-esterified fatty acid (NEFA; 0.57 ±0.14 v 0.35 ±0.05 mM) and triglyceride (TG; 0.47 ±0.11 vs. 0.25 ±0.08 mmol/L) concentrations (all P<0.05). Muscle triglyceride (TGm; 72.3 ±14.7 vs. 38.9 ±6.2 mmol/kg d.m., P<0.05) and diacylglycerol (DAG; 96 ±28 vs. 42 ±8 pmol/mg d.m., P<0.05) content were elevated in LCR vs. HCR. Accompanying the greater lipid accretion in LCR was increased FAT/CD36 content (1014 ±80 vs. 781 ±70 AU, P<0.05) and reduced TG lipase activity (0.158 ±0.0125 vs. 0.274 ±0.018 mmol/min/kg d.m., P<0.05). Muscle glycogen, GLUT4 protein and basal phosphorylation states of AMPK-&#945;1, -&#945;2 and ACC were similar in LCR and HCR. In conclusion, rats with low intrinsic aerobic capacity demonstrate abnormalities in lipid-handling capacity. These disruptions may, in part, be responsible for the increased risk of metabolic disorders observed in this phenotype.