We examined the effects of a high-fat diet (HFD-CHO) versus a habitual diet, prior to carbohydrate (CHO)-loading on fuel metabolism and cycling time-trial (TT) performance. Five endurance-trained cyclists participated in two 14-day randomized cross-over trials during which subjects consumed either a HFD (> 65% MJ from fat) or their habitual diet (CTL) (30 +/- 5% MJ from fat) for 10 day, before ingesting a high-CHO diet (CHO-loading, CHO > 70% MJ) for 3 days. Trials consisted of a 150-min cycle at 70% of peak oxygen uptake (VáO2peak), followed immediately by a 20-km TT. One hour before each trial, cyclists ingested 400 ml of a 3.44% medium-chain triacylglycerol (MCT) solution, and during the trial, ingested 600 ml/hour of a 10% 14C-glucose + 3.44% MCT solution. The dietary treatments did not alter the subjects' weight, body fat, or lipid profile. There were also no changes in circulating glucose, lactate, free fatty acid (FFA), and b-hydroxybutyrate concentrations during exercise. However, mean serum glycerol concentrations were significantly higher (p < .01) in the HFD-CHO trial. The HFD-CHO diet increased total fat oxidation and reduced total CHO oxidation but did not alter plasma glucose oxidation during exercise. By contrast, the estimated rates of muscle glycogen and lactate oxidation were lower after the HFD-CHO diet. The HFD-CHO treatment was also associated with improved TT times (29.5 +/- 2.9 min vs. 30.9 +/- 3.4 min for HFD-CHO and CTL-CHO, p <.05). High-fat feeding for 10 days prior to CHO-loading was associated with an increased reliance on fat, a decreased reliance on muscle glycogen, and improved time trial performance after prolonged exercise.