Transition of primiparous heifers to the milking herd is a period with multiple stressors. The objective of these studies was to determine effects of parlour experience and prepartum milking (pre-milking) on behavioural and physiological indicators of stress after calving. Two experiments were conducted, one was in a free-stall housing confinement system and the second was in a modified grazing system. Forty-eight first-calf heifers were assigned to three treatments: control; experienced heifers taken through the parlour without milking; or pre-milk heifers milked for 3 weeks prior to estimated parturition. Blood was collected within 24 h of parturition and on days 3, 5, 7, 10 and 14 following parturition for cortisol and acute phase protein determination. In the grazing system, 20 heifers were assigned to a prepartum milked or control group as in the confinement system and behaviour observations included days −21, −14, −7, −5, −3 and −1 relative to calving and days 1, 3, 7, 9, 14, and 16 post-calving. Milk production was greatest for prepartum milked heifers in both housing systems. However, somatic cell score was reduced by prepartum milking only in the confinement system. Balking occurred least in parlour-experienced heifers. In confinement housing, shifting while in the parlour was the only behaviour that was greater at first milking in control heifers. Kicking was most frequent for parlour experienced heifers on day 2. Grazing system pre-milked heifers shifted more at their first milking (day −21) than did the controls at their first milking (day 1). Shifting within cow was greatest on day −21 compared with day −5 (P<0·05). Pre-milked heifers shifted more on day 1 post-calving than did the control heifers (P<0·05). These results showed that shifting was the most indicative behaviour of restlessness, was transient, and decreased by day 5 prior to calving. Cortisol and α1-acid glycoprotein concentrations were not different; however, haptoglobin increased for all treatments up to and including day 3 and haptoglobin concentrations of pre-milked heifers began to decrease by day 5 post-calving. Pre-milked heifers had lower haptoglobin concentrations than the control heifers and tended to have lower concentrations than experienced heifers on day 10 post partum. By day 14 post partum, all haptoglobin concentrations were <200 μg/ml, but the haptoglobin concentration of control heifers was greater than that of pre-milked and experienced heifers. These results showed that prepartum milking and parlour experience shorten some acute phase protein responses, but minimally affect early parlour behaviours.