Animals often display behavioural and physiological stress responses during routine veterinary and management procedures. These responses can be dangerous to the animals and humans and might lower welfare and performance. The effects of previous positive handling and of gentle interactions during rectal palpation and a sham insemination on the behaviour and heart rate of dairy cows were investigated. Two groups of 10 cows each were studied. One group received positive handling for four weeks in addition to routine handling by one handler. The second group only received routine handling by different caretakers. Following the handling period, each cow received a rectal palpation on four days under four different situations: cow is alone, with the handler, with a usual caretaker, or with an unknown person. Lower heart rates, less kicking, and the tendency for less restless behaviour were found among cows that had experienced the positive handling. Gentle interactions during the test also calmed the cows. However, the person involved in the gentle interactions caused different reactions. Gentling by the handler resulted in less restless behaviour, compared to gentling by the other two people. The authors concluded that previous positive handling and gentle interactions by the positive handler during stressful procedures, causes a reduction in stress reactions. The person involved in interactions makes a difference in the calming effect on the animals. These results show the importance of positive interactions with animals, as they improve welfare and decrease the risk of dangerous responses by the animals during stressful procedures.