Tear fluid cortisol as a marker of acute stress reactivity: A validation study against salivary measures

Assessing the activation of major stress axes is fundamental to stress research. While saliva and blood are wellestablished ways for quantifying stress biomarkers, each have their own methodological and practical limitations. Tear fluid (TF) is a promising, non-invasive and low-cost alternative, containing markers that reflect both local and systemic physiological processes. Yet, it remains unclear whether TF-based stress biomarkers respond to acute (experimentally-induced) stress and how they compare with established "gold standard" methods. We, therefore, examined the stress reactivity of TF-based cortisol and alpha-amylase, and compared it directly to estimates obtained from saliva. Exploratory TF aims included assessments of test-retest reliability and quantifying adrenaline's responsiveness to stress-induction. Healthy participants with no history of mental disorders (N = 56) were exposed to the Maastricht Acute Stress Test experimental (MASTEXP;n = 29) or control (MASTCTL; n = 27) condition. TF and saliva samples were collected across multiple time points, with saliva serving as the reference fluid. Tear cortisol increased significantly following stress-induction and showed a positive association with salivary cortisol, thus supporting its validity as a stress biomarker. In contrast, tear alpha-amylase exhibited high variability and no reliable stress-related responsivity. Interestingly, both biomarkers exhibited distinct temporal patterns in each fluid, with TF-derived estimates peaking faster. Tear cortisol also demonstrated acceptable test-retest reliability across sessions. While detectable, tear adrenaline showed no responsiveness to acute stress. These findings provide initial evidence that TF represents a viable alternative for assessing stress biomarkers, particularly cortisol. With further validation, TF-based biomarkers could complement current approaches in psychoneuroendocrinology research.