We evaluated how the behavioural and heart rate response of dairy cows and calves to mutual separation was affected by two factors: the age of the calf at separation (1,4 and 7 days) and the presence or absence of visual and auditory contact between the mother and her calf after separation. In total, 46 cow-calf pairs were randomly allocated to one of six treatments according to a 3 x 2 factorial design. The cows‘ behavioural response to separation was recorded at 0-1, 9-10, 25-26 and 50 and 51 h after separation. The calves‘ behavioural response was recorded during the 24 h after separation. The heart rate was measured 60 min before and 60 min after separation. Cows from the later separation treatments spent more time standing (p < 0.01), placed their heads outside the pen (p < 0.001), vocalised (p < 0.05) and sniffed both the air (p < 0.01) and other animals (p < 0.001) more frequently compared to cows separated from their calves on day 1. The behavioural response was also more intense when the cows could see and hear their calves: they placed their heads outside the pen (p < 0.001), vocalised (p < 0.001) and sniffed the air (p < 0.01) more often than the no contact cows. Heart rate increased in all cows immediately after separation and was not influenced by the calf's age or by contact with the calf. After separation, older calves stood and moved more (p < 0.001), placed their heads outside the pen more often (p < 0.01) and showed more explorative behaviour (i.e. sniffing walls and bedding, p < 0.01; licking walls, p < 0.05) than the calves separated on day 1. Furthermore, the calves with visual and auditory contact with their mothers tended to place their heads outside the pen more often (p < 0.1) than the calves from the no contact treatments. Heart rate increased rapidly in all calves after separation; however, the increase lasted longer in the later separated calves (p < 0.05). At 3 weeks of age, the calves separated on day 7 habituated more quickly to the novel situation (p < 0.001). Calves separated on day 4 were more likely to engage in social play than the calves.