Pupil size as a sensitive marker of acute stress dynamics.

The aim of this study was to investigate pupil size as a sensitive and immediate marker of acute stress, by analyzing its evolution in response to two different stress-inducing cognitive tasks aiming to elicit distinct stress responses: a traditional Stroop task and a socially evaluative competitive Stroop task. Thirty participants were randomly assigned to either the traditional (G-trad, N = 14) or the competitive (G-comp, N = 16) Stroop groups. Stress-related measures-including pupil dilation, heart rate variability (RMSSD and SDNN), respiratory rate, blood pressure, salivary cortisol and α-amylase levels, and perceived stress (visual analog scale)-were recorded at baseline and during the 20-min recovery period. In addition, pupillary, cardiac, and respiratory responses were continuously recorded throughout the stress-inducing task, while perceived stress was assessed between task blocks. Significant increases in pupil dilation and perceived stress were observed only in G-comp during the task. Salivary cortisol increased significantly in G-comp after the Stroop, while α-amylase increased in both groups, though more markedly in G-comp. In this group, both cortisol and α-amylase exhibited a delayed peak, occurring 10 min post-task, before partially declining, reflecting a transient but intense hormonal stress response. No significant difference between the groups was found for RMSSD, SDNN, respiratory rate and blood pressure. Overall, pupil dilation was the only physiological measure recorded during task performance that differentiated between the traditional and competitive conditions. By contrast, hormonal markers exhibited delayed responses, while the other physiological measures failed to discriminate between conditions, supporting pupillometry as a non-invasive, real-time indicator of acute stress dynamics.