We examined the effects of novelty/isolation stress on cows at milking and whether human contact reduced the stress. Holstein cows (n=18) were observed during three experimental milkings following a balanced order: (1) control (C) — milked in usual place; (2) novelty/isolation stress (S) — milked alone in an unfamiliar room; (3) human contact (HC) — milked in unfamiliar room and brushed by a familiar person. Behavior and heart rate during milking, milk yield and residual milk following oxytocin injections were recorded, and blood samples assayed for oxytocin and cortisol. Cows defecated/urinated and vocalized more and made more steps during milking in the unfamiliar room and human contact prevented this increase. Cows kicked and lifted their legs less in the unfamiliar room, but human contact did not affect this. Both plasma cortisol concentrations and heart rates were higher when cows were milked in the unfamiliar room. Although human contact reduced heart rates during the initial period of isolation, heart rates during milking and cortisol concentrations were not affected by human contact. Milk yield was lower, residual milk higher, and oxytocin during milking was lower in the unfamiliar room but this was not changed by human contact. Cows milked alone in an unfamiliar room showed signs of acute stress and gave less milk due to higher residual milk and reduced oxytocin secretion. Human contact reduced some behavioral signs of agitation and heart rate, but had no effect on milk yield or hormonal responses. For dairy cows, human contact may not be sufficiently comforting to be able to reduce the endocrine response to novelty/isolation stress.