The primary aim of this study was to compare the measured oxygen consumption (Measured-VO2) in a simulated futsal game (S-Game) with the estimated oxygen consumption (Estimated-VO2) through a regression equation between heart rate (HR) and oxygen consumption (VO2) (HR-VO2) 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-VO2 was determined individually in the continuous test on the treadmill (ContTest). The Measured-VO2 in S-Game was compared with the Estimated-VO2 in the ContTest. Alactic and lactic pathways were estimated by VO2. The Estimated-VO2 presented no statistically significant difference with the Measured-VO2, using the paired t-test (p = 0.38). However, the correlation between Estimated- and Measured-VO2 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-VO2 determined by the ContTest was not a good individual predictor of VO2. The high intensity and intermittent nature of the futsal game possibly caused dissociation in the HR-VO2 relationship. ContTest is not recommended for estimating VO2 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%).