Circulating cortisol levels are accepted as a sensitive indicator of acute stress in marine mammals, particularly in relation with capture and handling. The present study provides the first long-term monitoring of cortisol levels in four harbour porpoises held in human care—an adult male and adult female and two juvenile females. It also compares levels in blood obtained after removing the animal from the water (OWR sampling) with levels in blood obtained at poolside under voluntary husbandry behaviours (VHB sampling). Cortisol levels differed significantly between the four porpoises, although they all exhibited quite high variations in cortisol levels, with averages of 64.9 and 70.5 μg/l in the adult male and female, respectively, and 90.7 and 51.4 μg/l in the juvenile females. OWR sampling induced significantly higher cortisol levels than VHB sampling, with a dramatic threefold decrease in circulating cortisol levels obtained under VHB sampling compared to levels obtained under OWR sampling (16.6 and 20.2 μg/l compared with 64.9 and 70.5 μg/l in the adult male and female respectively). Even if the porpoises showed some habituation to handling, regular and frequent handling over several years did not suppress a significant stress response in the porpoises when they were removed from the water, pointing to the advantage of using VHB for limiting stress in husbandry practices.