Resting and exercised (both acute and chronic) hindlimb skeletal muscle from long-lived Ames dwarf and wild type mice at 3, 12, 18, and 24 months of age was tested for antioxidant enzyme activity and protein, non-enzymatic antioxidant ratios, mitochondrial hydrogen peroxide concentration, and plasma lactate levels. Differences were observed in GPX enzyme activity between mouse genotypes at all physical activity levels, with dwarf mice exhibiting depressed levels at younger ages (3 months: P = 0.09 [non-swim], P = 0.03 [acute swim], P = 0.04 [chronic swim]) and comparatively higher levels than wild type mice at older ages (18-24 months: P = 0.05 [acute swim], P = 0.07 [chronic swim]). Catalase enzyme activity and the GSH system rarely demonstrated significant differences between genotypes, regardless of age or activity. However, the chronic exercise group displayed a difference in GSH:GSSG ratios between mouse genotypes (P = 0.005). Plasma lactate concentrations were elevated in the wild type mice compared to the dwarf mice at all ages in all activity groups. These results suggest there are biological differences with regard to antioxidant defense that favor the Ames dwarf mouse in active and resting skeletal muscle when compared to wild type mice.