Purpose: To analyze the diurnal variation of maximal fat oxidation (MFO) and the intensity that elicits MFO (Fat(max)) in trained male athletes. Methods: A total of 12 endurance-trained male athletes age 24.7 (4.1) y participated in the study. The authors measured MFO, Fat(max), maximum oxygen uptake (VO(2)max), and VO2 percentage at ventilatory threshold 2 with a graded exercise protocol performed on 2 days separated by 1 wk. One test was performed in the morning and the other in the afternoon. The authors assessed the participants’ chronotype using the HOME questionnaire. Results: MFO and Fat(max) were greater in the afternoon than in the morning (Delta = 13%, P < .001 and Delta = 6%, P = .001, respectively), whereas there were similar VO(2)max and ventilatory threshold 2 in the morning, than in the afternoon test (Delta = 0.2%, P = .158 and Delta = 7%, P = .650, respectively). There was a strong positive association between VO(2)max and MFO in both morning and afternoon assessments (R-2 = .783, P = .001 and R-2 = .663, P < .001, respectively). Similarly, there was a positive association between VO(2)max and Fat(max) in both morning and afternoon assessments (R-2 = .406, P = .024 and R-2 = .414, P = .026, respectively). Conclusion: MFO and Fat(max) may partially explain some of the observed diurnal variation in the performance of endurance sports.