This study evaluated the ability to use the relationship between heart rate (HR) and oxygen uptake (VO2) to estimate energy expenditure during physical activity (AEE). General prediction equations were established based on the individual relations between HR and AEE. Forty-two women, (mean age 38.1 [SD 9.8] years, BMI 23.9 [SD 4.4]), and 45 men (40.3 [SD 9.2] years, BMI 24.7 [SD 2.9]), carried out two incremental tests, one with a cycle ergometer and another on a treadmill. Subjects also performed a 10 minute steady-state exercise, cycling and walking. Respiratory gases were obtained from indirect calorimetry. AEE was calculated from VO2 and carbon dioxide production (VCO2). The predicted AEE was compared with the AEE measured during the steady-state exercise. Using the generalised linear model, two alternative models were found to predict AEE and HR. The first showed a three-way interaction between HR, body weight and gender (p = 0.022) and also between HR age and gender (p=0.083). The second produced a three-way interaction between HR, body weight and gender (p=0.057). For cycling and walking, respectively, the predicted AEE overestimated the actual AEE by 17.7 (SD 23.2)% and 6.2 (SD 19.3)% in the first model and by 17.9 (SD 22.7)% and 6.6 (SD 19.8)% in the second during the steady-state exercise. It was concluded that at least gender and body weight should be included when HR is used to predict AEE.