The accuracy of the American College of Sports Medicine (ACSM) equations for producing predicted values of heart rate reserve (HRR) and oxygen uptake reserve (VO2R) and consequently, target energy expenditure (EE) during exercise are yet to be established. This study investigated whether speeds defined by the ACSM metabolic equation for running correctly estimate the EEs during isocaloric exercise bouts. Twenty-eight men performed a ramp-incremental maximal exercise test to determine HRmax and VO2max. Two continuous exercise bouts at 60 and 80% VO2R and target EE of 400 kcal were then performed. In the 60% VO2R exercise the observed VO2R and EE were lower than predicted only during the first time quartile of the bout (t = 6.5, p < 0.001), whereas at 80% VO2R it was lower during the first (t = 15.3, p < 0.001), second (t = 5.4, p < 0.001) and third (t = 3.1, p = 0.025) quartiles. The observed HR was lower than predicted in the first time quartile of the 60% [Formula: see text] (t = 5.6, p < 0.001) and 80% VO2R bouts (t = 10.7, p < 0.001), whereas no significant differences occurred for any other time quartiles (p ≥ 0.23). In conclusion, the running speed defined by the ACSM metabolic equation overestimated VO2R and EE within exercise performed at 60 and 80% VO2R especially in higher intensities.