We studied the effects of a 38-day endurance exercise training program on leucine turnover and substrate metabolism during a 90-min exercise bout at 60% peak O2consumption (V˙o2 peak) in 6 males and 6 females. Subjects were studied at both the same absolute (ABS) and relative (REL) exercise intensities posttraining. Training resulted in a significant increase in whole bodyV˙o2 peakand skeletal muscle citrate synthase (CS; P < 0.001), complex I-III ( P < 0.05), and total branched-chain 2-oxoacid dehydrogenase (BCOAD; P < 0.001) activities. Leucine oxidation increased during exercise for the pretraining trial (PRE, P < 0.001); however, there was no increase for either the ABS or REL posttraining trial. Leucine oxidation was significantly lower for females at all time points during rest and exercise ( P < 0.01). The percentage of BCOAD in the activated state was significantly increased after exercise for both the PRE and REL exercise trials, with the increase in PRE being greater ( P < 0.001) compared with REL ( P < 0.05). Females oxidized proportionately more lipid and less carbohydrate during exercise compared with males. In conclusion, we found that 38 days of endurance exercise training significantly attenuated both leucine oxidation and BCOAD activation during 90 min of endurance exercise at 60%V˙o2 peakfor both ABS and REL exercise intensities. Furthermore, females oxidize proportionately more lipid and less carbohydrate compared with males during endurance exercise.