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/m²) 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 4^th min was significantly lower than that of the 2^nd and 3^rd min (p < 0.01). In addition, the 5^th and 6^th min fat oxidation rates were significantly lower than the rates of the 1^st, 2^nd, 3^rd, and 4^th min (0.30 ± 0.01 and 0.29 ± 0.01 g/min for the 5^th and 6^th min, respectively, vs. 0.35 ± 0.02, 0.34 ± 0.02, 0.33 ± 0.02, and 0.31 ± 0.01 g/min for the 1^st, 2^nd, 3^rd, and 4^th min, respectively; p < 0.01). Most of the participants had MFO rates in the 1^st min of the stage (16/35 participants), and the MFO rates of the remaining participants were observed in the 2^nd, 3^rd, and 4^th min (7/35, 4/35, and 8/35 participants, respectively). None of the participants had MFO rates in the 5^th or 6^th 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.

• Taking the average of the last 2 min to calculate the fat oxidation rate could risk underestimating the actual MFO rate.

• Individual maximal fat oxidation rates might be observed at different times in the Fatmax stage.

• Selecting an individual’s maximal fat oxidation rate rather than the average over a time period might give better results.