In order to assess relative thermal sensitivity between the day and the night, vaginal temperature (Tv), heat production (HP), heart rate (HR), respiration rate (RR), skin surface temperatures (from which mean skin temperature (Ts) was calculated) and standing time were measured at environmental temperatures (Te) of 23, 28, 33 and 38°C during the day (11.00–15.00 h) and during the night (23.00–03.00 h) using four Holstein heifers. Both Tv and mean body temperature (Tb) were greater during the night than during the day, increased with increased Te, and the rate of increase of both Tv and Tb with increased Te was greater during the night (P<0·05). Estimated mean HP was similar during the night and during the day, and HP did not increase with increased Te. Respiration rate was greater but not significantly different at night compared to during the day, and increased with increased Te. Mean skin temperature was similar between day and night, and increased with increased Te. The amount of time engaged in standing activity was greater but not significantly different during the night than during the day and standing activity increased with increased Te. The results suggest that thermal sensitivity is lower during the night than during the day, and consequently the greater night responses of Tv and Tb, over day responses, are a requirement for the maintenance of heat balance.