Purpose: To analyze the diurnal variation of maximal fat oxidation (MFO) and the intensity that elicits MFO (Fatmax) 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, Fatmax, maximum oxygen uptake (VO2max), 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 HÖME questionnaire. Results: MFO and Fatmax were greater in the afternoon than in the morning (Δ = 13%, P < .001 and Δ = 6%, P = .001, respectively), whereas there were similar VO2max and ventilatory threshold 2 in the morning, than in the afternoon test (Δ = 0.2%, P = .158 and Δ = 7%, P = .650, respectively). There was a strong positive association between VO2max 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 VO2max and Fatmax in both morning and afternoon assessments (R 2 = .406, P = .024 and R 2 = .414, P = .026, respectively). Conclusion: MFO and Fatmax may partially explain some of the observed diurnal variation in the performance of endurance sports.