The growing prevalence of obesity and metabolic disorders such as type II diabetes, is associated with reduced exercise and maximal fat oxidation rate (Fatmax). The aim of this study was to verify the reliability of a new test protocol (Fatmax_work) using the INCA software package. Twenty four sedentary subjects was enrolled and performed the following tests using a VO2000 metabolimeter: (1) An incremental test of 20 min to determine the individual Fatmax and maximum rate of fat oxidation (MFO) with new Fatmax_work test; (2) a constant load test of 60 min. For our subjects, the average fat zone was estimated by INCA at 54.2 ± 4.9% max HR, RER 0.86 ± 0.09, 130 ± 64 W. The results from the constant load test differed by 0.01 ± 1.92 for respiratory gas exchange rate (RER) and -0.22 ± 1.92% for heart rate (HR). This study showed that the Fatmax_work test can be used to predict Fatmax in untrained overweight subjects and INCA is a reliable and accurate software package.