Purpose: The present study examined the effects of endurance training on the contractile and biochemical properties of the rat costal diaphragm in vitro. Methods: Sixty-four rats were divided into two groups: exercise trained (T) and control (C). Training consisted of treadmill running 5 d wk(-1), 60 min.d(-1) at similar to 70% of (V) over dotO(2max), over a 10-wk period. Results: Control diaphragm strips produced an average of 12% less force from minute 15 to 50 of a 60-min in vitro fatigue protocol, compared with the T diaphragm strips (P < 0.01). T diaphragms had 10.1% higher citrate synthase (CS) and 12.1% higher superoxide dismutase (SOD) activities compared with the C (P < 0.05). Despite a significant decrease (P < 0.05) in Type IIb myosin heavy chains (MHC) and an increase (P < 0.05) in Type I MHC in T diaphragms, maximal shortening velocity (V-max) in the diaphragm was not different between T and C animals. No differences were observed in specific force or the relative proportions of myosin light chains between groups. Conclusions: These findings suggest that endurance training reduces the rate of diaphragm fatigue in vitro but has no effect on V-max or specific force.