The primary aim of this study was to compare the measured oxygen consumption (Measured -V O2) in a simulated futsal game (S-Game) with the estimated oxygen consumption (Estimated -V O2) through a regression equation between heart rate (HR) and oxygen consumption ( V O2) (HR- V O2) in treadmill running, and a secondary aim was to calculate the total energy expenditure (EE) in S-Game. Ten professional players (22.20 ± 3.22 years) were evaluated. HR- V O2 was determined individually in the continuous test on the treadmill (ContTest). The Measured -V O2 in S-Game was compared with the Estimated -V O2 in the ContTest. Alactic and lactic pathways were estimated by V O2. The Estimated -V O2 presented no statistically significant difference with the Measured -V O2, using the paired t -test ( p = 0.38). However, the correlation between Estimated- and Measured- V O2 was very weak ( r = −0.05), and it presented poor agreement (concordance correlation coefficient = −0.04). In addition, a Bland–Altman plot presented bias of −2.8 ml/kg/min and individual difference as large as 19 ml/kg/min. The HR- V O2 determined by the ContTest was not a good individual predictor of V O2. The high intensity and intermittent nature of the futsal game possibly caused dissociation in the HR- V O2 relationship. ContTest is not recommended for estimating V O2 and calculating individual EE in the futsal game. This is recommended only for the group mean. The total EE in S-Game was 13.10 ± 1.25 kcal.min−1 (10.81 ± 1.57 metabolic equivalents). The contributions from the metabolic pathways were as follows: aerobic (93%), alactic (5%), and lactic (2%).