The main purpose of the present study was to investigate the association between perceived stress and impaired cutaneous wound healing in humans using a novel wound assessment technique, and taking into account putative mediating factors such as cortisol levels, health behaviours, and personality factors. The study made use of a prospective, within-subjects design in which 24 male non-smokers participated. Every subject received a standard 4mm-punch biopsy, and the healing progress was monitored via high-resolution ultrasound scanning. Participants completed questionnaires on perceived stress, health behaviours, and personality factors, and sampled saliva for cortisol assessment after awakening at 2 weeks prior, directly after, and 2 weeks after the biopsy. The overall results showed a significant negative correlation between speed of wound healing, and both Perceived Stress scale (PSS) scores (r=−.59; p<.01), and General Health Questionnaire (GHQ) scores (r=−.59; p<.01) at the time of the biopsy. The area under the morning cortisol response curve was negatively correlated with speed of wound healing (r=−.55; p<.05), indicating a clear elevation in the morning cortisol slope of those whose wounds were slowest to heal. A median split of the complete sample yielded that the ‘slow healing’ group showed higher stress levels (PSS t=3.93, p<.01, GHQ t=2.50, p<.05), lower trait optimism (t=3.25, p<.05), and higher cortisol levels to awakening (F=5.60, p<.05) compared with the ‘fast healing’ group. None of the health behaviours investigated (i.e. alcohol consumption, exercise, healthy eating, and sleep) were correlated with healing speed at any time point. Our data hint at a considerable influence of stress on wound healing, and suggests that elevated cortisol levels, rather than altered health behaviours, play a role in this effect.