Fat oxidation during exercise can be influenced by exercise intensity and duration, training status, diet and physical activity level. However, few studies have investigated the effect of time of day on the rate of fat oxidation during exercise. Thus the present study was designed to investigate the effect of time of day on indexes of fat oxidation in active young women. For this purpose, 14 healthy women (mean±SD age, BF%, weight, BMI and Vo2max: 21.6±1.6 years, 18.3± 4.6 %, 51.2± 3.3 kg, 22±1.7 kg/m2, 40.8±4.2 ml/kg.min) voluntarily participated in this study. The exercise test was performed in two separate sessions in the morning (7:00) and evening (19:00). There was a 7-day interval between the two tests. The protocol was an incremental exercise to exhaustion on a treadmill. The volunteers started exercising at a speed of 58.3 m/min and at a gradient of 1%. Then, the speed increased to 16.7 m/min every three minutes until 125 m/min. At this point, the gradient increased by 2% per three minutes until RER=1 was achieved. VO2 and VCO2 were measured throughout the exercise by an automated gas analysis system. The data were analyzed using independent t test (?=0.05). The findings of the present study showed that no significant difference in maximal fat oxidation between morning and evening exercises (morning: 0.39±0.13 vs. evening: 0.44±0.11, P>0.05). There was no significant difference in FAT Max between morning and evening (P>0.05). However, the mean intensity of maximal fat oxidation was higher in the evening. Finally, it can be concluded that probably time of day (morning and evening) does not have an effect on substrate oxidation during intensive exercise.