A linear relationship between heart rate (HR) and oxygen consumption (VO2) has been reported in homeothermic animals, indicating that is possible to estimate heat production through HR measurements. This relationship may depend on the animal activity and environmental conditions. The main objective of the present study was to evaluate the effect of the air temperature and animal posture and activity on heat production and VO2 in relation to HR. In addition, as a secondary objective, the energy cost of eating and ruminating versus idling and standing versus lying down was determined. Twelve Holstein lactating cows were housed inside climate-controlled respiration chambers for 8 d, where the air temperature was gradually increased from 7 to 21°C during the night and from 16 to 30°C during the day with daily increments of 2°C for both daytime and nighttime. During the 8-d data collection period, HR and gaseous exchange measurements were performed, and animal posture and activity were recorded continuously. The oxygen pulse (O2P), which represents the amount of oxygen that is consumed by the cow per heartbeat, was calculated as the ratio between VO2 and HR. Results showed that heat production and VO2 were linearly and positively associated with HR, but this relationship largely varied between individual cows. Within the range tested, O2P was unaffected by temperature, but we detected a tendency for an interaction of O2P with the temperature range tested during the night versus during the day. This indicates that the effect of air temperature on O2P is nonlinear. Standing and eating slightly increased O2P (1.0 and 2.5%) compared with lying down and idling, respectively, whereas rumination increased O2P by 5.1% compared with idling. It was concluded that the potential bias introduced by these effects on the O2P for the application of the technique is limited. The energy cost of eating and ruminating over idling was 223 ± 11 and 45 ± 6 kJ/kg0.75 per day, respectively, whereas the energy cost of standing over lying down was 53 ± 6 kJ/kg0.75 per day. We concluded that O2P in dairy cows was slightly affected by both animal posture and activity, but remained unaffected by air temperature within 8 to 32°C. Nonlinearity of the relationship between the O2P and air temperature suggests that caution is required extrapolating O2P beyond the temperature range evaluated in our experiment.