Differences in physiological functioning in the hypothalamus-pituitary-adrenal cortex axis and the autonomic nervous system have been suggested to contribute to many of the health differences that may be observed between men and women as well as being relevant for the development of musculoskeletal pain. To clarify whether men and women with identical work tasks reacted differently when doing work known to induce musculoskeletal pain, ratings and physiological measurements were obtained at work start (15:30) and at the end of the workshift (22:30) on 17 men and 20 women. Men showed a larger decrease in perceived energy levels during the workshift but there were no differences between men and women as regards cortisol, adrenaline, noradrenaline, heart-rate activation, perceived stress, pain and physical exertion. In conclusion, differential physiological activation during the workshift seem to be an unlikely mechanism for explaining gender differences in pain associated with exposure to awkward and repetitive movements.