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 well-established 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 (MAST_EXP;n = 29) or control (MAST_CTL;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.