•A refined virtual reality (VR) adaptation of the Trier Social Stress Test (TSST) was tested against the standard in vivo TSST.•Stress-induced increases of free salivary cortisol and alpha amylase were comparable in both stress groups compared to control groups.•The mean subjective feelings of stress did not differ between both stress groups but were higher than in the control groups.•The VR adaptation of the TSST has the potential to induce robust physiological and subjective stress reactions. In recent years, virtual reality (VR) technology has found its way into nearly all fields of psychology. Previous studies indicated that virtual reality adaptations of the TSST are less potent in stimulating HPA-axis responses, with lower salivary cortisol responses recorded as compared to the in-vivo TSST. (TSST-IV). In the present experiment we tested the stress-induction potential of a refined version of the TSST-VR using a fully orthogonal experimental design in which ninety-three healthy males were either assigned to the TSST condition or a corresponding control condition in a real or virtual environment. We found a significant increase of endocrine, autonomic and self-reported stress markers in both stress conditions. Notably, we found a robust rise in salivary cortisol to the TSST-VR comparable to that observed in the TSST-IV. Despite subtle differences in response between virtual and in vivo settings, we conclude that VR adaptations of in-vivo stressors have the potential to induce real physiological and subjective reactions.