Abstract: Veterinary and management procedures often are aversive to the animals, resulting in physiological and behavioural stress reactions, which increase the risk of accidents and might lower performance. We investigated the effects of previous positive handling and of gentle interactions during the procedure on behaviour and heart rate in dairy cows during rectal palpation with sham insemination. Twenty cows were allocated randomly into two groups of 10 animals: handling, received additional positive handling over a period of four weeks by one person (handler); control, only routine handling by different caretakers. The week after the handling period, tests lasting 9 min, including 4 min rectal palpation, were carried out with each animal on four successive days in four situations in a balanced order: cow is alone during the test, with the handler, with an usual caretaker, or with an unknown person. Behaviour and heart rate were recorded. Previously handled animals had lower heart rate during tests (P<=0.05, n=19), kicked less when alone (P<=0.05, n=19) and tended to show less restless behaviour (P<=0.1, n=19). Cows were further calmed by gentle interactions during the test, but people differed remarkably. Cows showed less restless behaviour when gentled by the handler, both in the 4 min of rectal palpation and in the 9 min test period (each: P<=0.001, n=19). No significant stress reducing effect was found for the other two persons. In conclusion, stress reactions of cows during rectal palpation/insemination can be reduced by previous positive handling as well as by a person providing positive, gentle interactions during the procedure. The results underline the importance of positive, gentle interactions with the animals to enhance animal welfare and reduce the risk of accidents. They also show that people differ in the success to calm down the animals in aversive situations and indicate the need to investigate the characteristics responsible for the differences and identify the preconditions for a stress reducing effect in future research.