Our objective was to determine the influence of a high-fat diet (HFD) on fasting and postprandial skeletal muscle substrate metabolism in endurance-trained (ET) compared with sedentary (SED) humans. SED (n = 17) and ET (n = 7) males were control-fed a 10-day moderate-fat diet followed by a 5-day isocaloric HFD (55% fat. 30% carbohydrate). Skeletal muscle biopsies were taken in the fasted condition and 4 h after a high-fat meal (820 kcals; 63% fat and 25% carbohydrate). Palmitate-induced suppression of pyruvate oxidation, an indication of substrate preference. and oxidation of fat and glucose were measured in homogenized skeletal muscle in fasted and fed states. Postprandial responses were calculated as percent changes from fasting to fed st...