Cardiopulmonary fitness assessment is a valuable resource to obtain quantitative indicators of an individual's physical performance. The cardiopulmonary exercise test (CPX), considered the gold standard test for this evaluation, is costly and difficult to be accessed by the general population. In order to make this evaluation more accessible, and to better reflect the performance of daily life activities, alternative tests were proposed. Morbidly obese patients present limitations that impair physical performance assessment and could benefit from a test of shorter duration, provided it is validated. This observational study aimed to validate the two-minute step test (2MST) as a tool to evaluate functional capacity (FC) in obese with comorbidities and morbidly obese patients, compared the 2MST with CPX as a measure of physical performance, and developed a predictive equation to estimate peak oxygen uptake (VO2) in the 2MST. The CPX and the 2MST were performed and metabolic and ventilatory parameters were recorded in 31 obese individuals (BMI>35 kg/m2). Pearson correlation and multiple linear regression analyses were performed to evaluate the peak VO2 best predictors. Bland-Altman analysis was performed to assess the agreement between the two methods. Peak VO2 measured by CPX and 2MST showed a strong correlation (r=0.70, P<0.001) and there was a moderate correlation between peak VO2 of the 2MST and the number of up-and-down step cycles (UDS) (r=0.55; P=0.01). The reference equation obtained was: VO2 (mL·kg-1·min-1) = 13.341 + 0.138 × total UDS - (0.183 × BMI), with an estimated standard error of 1.3 mL·kg-1·min-1. The 2MST is a viable, practical, and easily accessible test for FC. UDS and BMI can predict peak VO2 satisfactorily.