This study aimed to compare the maximal fat oxidation (MFO) rates obtained from the stage average, last 2 min average, and highest value in the Fatmax stage determined with a 6 min step protocol. A total of 35 overweight, sedentary healthy men (age: 25.4 +/- 0.7 years, body mass index: 26.0 +/- 0.6 kg/m(2)) participated in the study. Substrate oxidation was calculated using breath-by-breath gas exchange data for each stage. When the change in the fat oxidation rate for every min throughout the Fatmax stage was evaluated, the average value of the 4th min was significantly lower than that of the 2nd and 3rd min (p < 0.01). In addition, the 5th and 6th min fat oxidation rates were significantly lower than the rates of the 1st, 2nd, 3rd, and 4th min (0.30 +/- 0.01 and 0.29 +/- 0.01 g/min for the 5th and 6th min, respectively, vs. 0.35 +/- 0.02, 0.34 +/- 0.02, 0.33 +/- 0.02, and 0.31 +/- 0.01 g/min for the 1st, 2nd, 3rd, and 4th min, respectively; p < 0.01). Most of the participants had MFO rates in the 1st min of the stage (16/35 participants), and the MFO rates of the remaining participants were observed in the 2nd, 3rd, and 4th min (7/35, 4/35, and 8/35 participants, respectively). None of the participants had MFO rates in the 5th or 6th min. The individual MFO rate (highest fat oxidation rate during Fatmax) was significantly higher than the fat oxidation rate calculated with the last 2 min average values (0.36 +/- 0.02 and 0.30 +/- 0.01 g/min, respectively; p < 0.05). In conclusion, the calculation of the fat oxidation rate by averaging the last portion of the Fatmax stage data may cause the underestimation of the MFO rate, which probably occurs earlier in the Fatmax stage.