BackgroundThere is evidence that caffeine increases the maximal fat oxidation rate (MFO) and aerobic capacity, which are known to be lower in the morning than in the afternoon. This paper examines the effect of caffeine intake on the diurnal variation of MFO during a graded exercise test in active men.MethodsUsing a triple-blind, placebo-controlled, crossover experimental design, 15 active caffeine-naive men (age: 327years) completed a graded exercise test four times at seven-day intervals. The subjects ingested 3mg/kg of caffeine or a placebo at 8am in the morning and 5pm in the afternoon (each subject completed tests under all four conditions in a random order). A graded cycling test was performed. MFO and maximum oxygen uptake (VO2max) were measured by indirect calorimetry, and the intensity of exercise that elicited MFO (Fat(max)) calculated.ResultsMFO, Fat(max) and VO2max were significantly higher in the afternoon than in the morning (all P<0.05). Compared to the placebo, caffeine increased mean MFO by 10.7% (0.280.10 vs. 0.31<plus/minus>0.09g/min respectively, P<0.001) in the morning, and by a mean 29.0% (0.31<plus/minus>0.09 vs. 0.40 +/- 0.10g/min, P<0.001) in the afternoon. Caffeine also increased mean Fat(max) by 11.1% (36.9<plus/minus>14.4 [placebo] vs. 41.0 +/- 13.1%, P=0.005) in the morning, and by 13.1% (42.0 +/- 11.6 vs. 47.5 +/- 10.8%, P=0.008) in the afternoon.Conclusion These findings confirm the previously reported diurnal variation in the whole-body fat oxidation rate during graded exercise in active caffeine-naive men, and indicate that the acute ingestion of 3mg/kg of caffeine increases MFO, Fat(max) and VO2max independent of the time of day.Trial registration NCT04320446. Registered 25 March 2020 - Retrospectively registered