We investigated the effect of endurance training on whole body substrate, glucose, and glycerol utilization during 90 min of exercise at 60% peak O2 consumption (V˙o 2 peak) in males and females. Substrate oxidation was determined before and after 7 wk of endurance training on a cycle ergometer, with posttesting performed at the same absolute (ABS, W) and relative (REL, %V˙o 2 peak) intensities. [6,6-2H]glucose and [1,1,2,3,3-2H]glycerol tracers were used to calculate the respective substrate tracee flux. Endurance training resulted in an increase inV˙o 2 peak for both males and females of 17 and 22%, respectively ( P < 0.001). Females demonstrated a lower respiratory exchange ratio (RER) both pretraining and posttraining compared with males during exercise ( P< 0.001). Glucose rate of appearance (Ra) and rate of disappearance (Rd) were not different between males and females. Glucose metabolic clearance rate (MCR) was lower at 75 and 90 min of exercise for females compared with males ( P < 0.05). Glucose Ra and Rd were lower during exercise at both ABS and REL posttraining exercise intensities compared with pretraining ( P < 0.001). Females had a higher exercise glycerol Ra and Rd compared with males both pre- and posttraining ( P < 0.001). Glycerol Ra was not different at either the ABS or REL posttraining exercise intensities compared with pretraining. We concluded that females oxidize proportionately more lipid and less carbohydrate during exercise compared with males both pre- and posttraining, which was cotemporal with a higher glycerol Ra in females. Furthermore, endurance training resulted in a decrease in glucose flux at both ABS and REL exercise intensities after endurance exercise training.