# Validating the Threat of a Virtual Reality Clinical Environment: A Mixed Methods Study
## 4 Discussion
Past VR studies of threat have employed more stressful or threatening stimuli, for instance, a fear of heights [41], or socio-evalative contexts such as the Trier Social Stress Test (TSST, a psychosocial stressor shown to elevate acute cortisol levels [42]). The VR conditions in the current study were intended to be realistic clinical settings and, as such, may not have been adequately provocative for a physiological reaction in the hypothalamic-pituitary-adrenal (HPA) axis. However, it is notable that there was no change in cortisol concentration after exposure to the non-threatening condition, which we designed to be comparable apart from the threat value itself. This leads us to propose that there was an effect on the HPA axis release of cortisol, even if the direction was unexpected or at least difficult to explain. It is possible that the timepoints chosen for post-VR cortisol sampling did not capture the expected stress response. Repeated measures of salivary cortisol are certainly advised to profile the concentration [43]. We based our testing times on a cortisol study that used a VR height challenge [13] and on an overview of the determinants of salivary cortisol responses to acute stress, although that review did recommend measuring at 15 to 20 min post-exposure [44]; hence, it is possible that the cortisol concentration in the threat condition peaked before 20 min. It has been recommended that to investigate the reactivity of the HPA axis to an acute stimulus, saliva should be collected before the stressor and at 15, 30, 45 and 60 min post- stressor. This allows for an evaluation not only of the stress response but also of recovery [45]. There is evidence from animal research to suggest that a rapid uptake of cortisol into cells could explain the apparent decrease we observed in the current investigation [46]. It is possible that 20 min after exposure was too late to capture marked increases in the cortisol concentration; if intracellular uptake occurred prior to sampling, no acute increase would be detected. Salivary cortisol concentrations reflect free plasma cortisol and cannot account for the fraction that is bound or stored intracellularly [46; 47]. Interestingly, our findings somewhat align with those of an investigation of reported stress and salivary cortisol concentrations in students under (natural) examination stress. That study also reported that salivary cortisol and perceived stress were inversely correlated, indicating that the relationship is not altogether clear [48]. The cortisol reduction we observed in the threat condition is unlikely to represent the expected decrease in cortisol according to the natural circadian rhythm [49] or potentially elevated stress levels in subjects on arrival to their study session (i.e., at cortisol); if either of these explained the reduction, we would have observed a cortisol change in both groups. It is a strength of the study design that subjects were randomised into the two groups and were tested in the same physical environment, so it is doubtful that a hormonal difference existed at baseline. It is hard to argue with the subjects' own reported feelings within the VR context, even if the 2 QT used was not a validated questionnaire. The significant difference between the groups in the subjective 2 QT responses lends strength to the study findings, as does the demonstrated internal consistency of the scale. Some limitations of the current work need to be acknowledged. We did not meet our projected sample size and, thus, it is possible we were underpowered to detect between-group differences in cortisol levels. The findings from the current sample, predominantly people aged around 25 years, may not be generalisable to an older population [50], though this has come under debate [51; 52]. We would have more closely controlled for confounders with a within-subject (counter-balanced or randomised) design. However, it would be challenging to facilitate such study sessions.