The aim of this study was to evaluate the stress suffered by medical residents as the result of being on call for 24 hours, from a multidimensional approach. Two groups of medical residents selected according to their work shift, participated in the study: one group (n = 40) was sleep-deprived after having been actively on-call for 24 hours, and another contrast group (n = 18) had performed a normal work day and were not sleep-deprived. All participants completed pre-post measures during a 24 h cycle. These were administered on both occasions at 8 am. The measures included HRV, cortisol, cognitive performance and transitory mood. The effect of the group x phase interaction was significant for all variables analysed, indicating that doctors in the 24h on-call shift group showed significant deterioration in all physiological, performance and mood indicators in comparison with the participants in the group not on call. These results suggest the need to review medical on-call systems, in order to reduce the stress load, which has a direct effect on working conditions.