The effect of intramyocellular lipids (IMCLs) on endurance performance with high skeletal muscle glycogen availability remains unclear. Previous work has shown that a lipid-supplemented high-carbohydrate (CHO) diet increases IMCLs while permitting normal glycogen loading. The aim of this study was to assess the effect of fat supplementation on fat oxidation (F-ox) and endurance performance. Twenty-two trained male cyclists performed 2 simulated time trials (TT) in a randomized crossover design. Subjects cycled at similar to 53% maximal voluntary external power for 2 h and then followed 1 of 2 diets for 2.5 days: a high-CHO low-fat (HC) diet, consisting of CHO 7.4 g.kg(-1).day(-1) and fat 0.5 g.kg(-1).day(-1); or a high-CHO fat-supplemented (HCF) diet, which was a replication of the HC diet with similar to 240 g surplus fat (30% saturation) distributed over the last 4 meals of the diet period. On trial morning, fasting blood was sampled and F-ox was measured during an incremental exercise; a similar to 1-h TT followed. Breath volatile compounds (VOCs) were measured at 3 time points. Mental fatigue, measured as reaction time, was evaluated during the TT. Plasma free fatty acid concentration was 50% lower after the HCF diet (p < 0.0001), and breath acetone was reduced (p < 0.05) "at rest". F-ox peaked (similar to 0.35 g.kg(-1)) at similar to 42% peak oxygen consumption, and was not influenced by diet. Performance was not significantly different between the HCF and HC diets (3369 +/- 46 s vs 3398 +/- 48 s; p = 0.39), nor were reaction times to the attention task and VOCs (p = NS for both). In conclusion, the short-term intake of a lipid supplement in combination with a glycogen-loading diet designed to boost intramyocellular lipids while avoiding fat adaptation did not alter substrate oxidation during exercise or 1-hour cycling performance.