Rumenocentesis is commonly used to collect rumen fluid to screen for acidosis. This study was designed to investigate whether rumenocentesis induced pain and stress and, if so, whether local anaesthesia could limit this. Twenty-four dairy cows were assigned to one of three treatments: (1) rumenocentesis with local anaesthesia (AR); (2) rumenocentesis without local anaesthesia (R); and (3) local anaesthesia only (A). Treatments were performed in a restraining cage. The cows were placed in the cage on three consecutive days and anaesthesia and/or nimenocentesis was performed on the second day. Blood samples for cortisol determination and heart rate were recorded from 0.25 h before treatment until 4 h after. Behaviour was noted while the cows were caged. Feed intake and milk production were measured the week before treatment, on the day of treatment, and the day after. With all three treatments, cortisol concentrations and heart rate were increased while cows were in the cage. Cortisol, cardiac and behavioural responses were not significantly higher in the R and AR treatments than the A group. Cortisol concentrations and heart rate did not change between days. Feed intake and milk production were unaffected by the treatments. It was concluded that rumenocentesis does not appear more stressful than local anaesthesia or handling.