Objective: Acute stress protocols are important tools for laboratory research on acute stress mechanisms. Several reasons necessitate varying body posture. However, the effect of such variations has not been systematically studied. We therefore examined the impact of body posture (sitting vs. standing) on biological and psychological stress responses induced by the Trier Social Stress Test (TSST) and its control condition (friendly TSST; f-TSST). Methods: One hundred four participants (57.69% female, mean age: 22.28, SD = 3.44) were randomly assigned to the sitting versus standing group and underwent the TSST and f-TSST on 2 separate days in randomized order. Stress responses were measured through salivary cortisol and alpha-amylase, HR, HRV, and self-reports. Results: Biological stress responses were higher in the TSST condition and the standing group (eg, maximum cortisol increase: p = .004). Psychological stress response was higher in the stress condition (eg, negative affect: p < .001), but was not different between posture groups (eg, negative affect: p = .819). No differences were observed in any markers after adjusting the stress-response measures for the non-stress condition (smallest p = .173). Conclusions: Although biological, but not psychological stress responses were affected by body posture overall, these effects did not persist when responses in the stress condition were adjusted by subtracting the non-stress values from the f-TSST. This implies that the effect of body posture can be neglected in studies with a non-stress control condition, but that its effects should be taken into account in studies with only the stress protocol.0