Endurance exercise modifies regulatory systems which control skeletal muscle Na⁺ and K⁺ fluxes, in particular Na⁺-K⁺-ATPase-mediated transport of these ions. Na⁺ and K⁺ ion channels also play important roles in the regulation of ionic movements, specifically mediating Na⁺ influx and K⁺ efflux that occurs during contractions resulting from action potential depolarization and repolarization. Whether exercise alters skeletal muscle electrophysiological properties controlled by these ion channels is unclear. The present study tested the hypothesis that endurance exercise modifies diaphragm action potential properties. Exercised rats spent eight weeks with free access to running wheels, and were compared with sedentary rats living in conventional rodent housing. Diaphragm muscle was subsequently removed under anesthesia, and studied in vitro. Resting membrane potential was not affected by endurance exercise. Muscle from exercised rats had a slower rate of action potential repolarization than that of sedentary animals (P = 0.0098), whereas rate of depolarization was similar in the two groups. The K⁺ channel blocker 3,4-diaminopyridine slowed action potential repolarization and increased action potential area of both exercised and sedentary muscle. However, these effects were significantly smaller in diaphragm from exercised than sedentary rats. These data indicate that voluntary running slows diaphragm action potential repolarization, most likely by modulating K⁺ channel number or function.