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The aim of the present study was to assess the effects of the ovum pick-up (OPU) technique on animal well-being. Eight dairy heifers were subjected to 4 months of twice-weekly OPU. The physiological response to OPU was recorded in four heifers at two sessions, at the beginning (time 1) and at the end (time 2) of the 4-month period. Heart rates were measured and blood was analysed for cortisol, vasopressin and PG-metabolite before, during (every 5 and 2inline image min), and after the OPU sessions. Reactions to each subprocedure of OPU (‘restraint’, ‘epidural’, ‘device in’ and ‘puncture’) were closely observed. In all heifers, reactions to the OPU procedures were also noted throughout the experimental period, and changes in routine behaviour, oestrous behaviour, body temperature, or other clinical traits were recorded. Subsequent to the experiment, the ovaries and tails were carefully inspected. At time 1, there was an insignificant increase in heart rate and cortisol throughout the OPU procedure. At time 2, these two parameters increased significantly, but both parameters declined to pre-OPU levels 10 min after completion of the procedure. No significant changes were seen in vasopressin or PG-metabolite at time 1 and time 2. Behaviourally, the heifers showed the strongest response to epidural anaesthesia, with a tendency for more intense response during the late 4-month sessions. The response to ‘device in’ and ‘puncture’ varied among individuals independently of time. There were no changes in the routine or oestrous behaviour throughout the experiment and no signs of clinical disorders. No major pathological changes were macroscopically seen in the ovaries and tails subsequent to the 4 months of OPU. In conclusion, the heifers showed a response to OPU, mostly to administration of epidural anaesthesia. However, we demonstrated that epidural anaesthesia can be administered in a way causing less discomfort.