BACKGROUND: Although the Trier Social Stress Test (TSST) constitutes a valid paradigm for social stress induction, less is known about the effects of a virtual reality (VR) TSST on short- and long-term hypothalamic-pituitary-adrenal (HPA) axis and sympathetic-adreno-medullar (SAM) axis responses. Hence, this study set out to evaluate reactivity and habituation of self-reported stress and HPA and SAM reactivity in a real TSST and VR-TSST when compared to a placebo TSST. METHOD: Sixty-eight healthy young adults (50% female) were randomly assigned to either a real TSST, a VR-TSST, or a placebo TSST, all of which were conducted three times (one day and one week post initial exposure). Social presence, self-reported stress, salivary cortisol, heart rate (HR), and heart rate variability (HRV) were analyzed using ANOVAs and multilevel models. FINDINGS: On the first exposure, both the real and VR-TSST showed significantly stronger cortisol and cardiovascular responses than the placebo. On the second visit, the cortisol response was still significantly high-and the HRV response low-for the real and VR-TSST. The third visit resulted in HR, HRV, and cortisol responses comparable to the placebo group. Furthermore, the real TSST induced more self-reported stress than the placebo on all three visits, the VR-TSST only on the first two visits. Social presence was stable across conditions and had no association with stress markers. CONCLUSION: These findings imply that the replicability of stress exposures at shorter intervals seems problematic for the traditional TSST, and for the VR-TSST.