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The Trier Social Stress Test (TSST) is a reliable and efficient protocol to induce acute psychosocial stress in the laboratory. If circumstances do not allow in-person assessments, an online version of the TSST could create more flexible research opportunities. To date, studies have confirmed subjective and autonomic stress responses to online TSST protocols. In this preregistered study (https://osf.io/u57aj), we focused on the effect of a TSST online for adults (TSST-OA) on cortisol and alpha amylase levels, and pleasure and arousal ratings. As cortisol stress reactivity is mediated by sex, we further compared men and women. We hypothesized significant increases in cortisol, alpha amylase and arousal, and a decrease in pleasure in response to the TSST-OA. Also, we expected stronger cortisol responses in males as compared with females, as in the laboratory TSST. N = 48 adults (56% female, mean_age=23.02 …