Stimulation of the vagus nerve influences biomarkers associated with both the hypothalamic–pituitary–adrenal (HPA) axis and the autonomic nervous system.The goal of the study was to evaluate the effect of transcutaneous vagus nerve stimulation (tVNS) on stress biomarkers and related symptoms and compare the effect of unilateral and bilateral stimulation.Total of 37 participants remained throughout the entire study. One-centimeter hair segments were taken and analyzed, representing cortisol and cortisone accumulation over the most recent month. The segments were taken before the study and after 4 weeks of stimulation. Three questionnaires (GAD-7, PHQ-9, PSQI) were used to evaluate anxiety and depressive symptoms as well as sleep quality during the study.Analysis of hair cortisol showed statistically significant reduction across the full sample (n = 37, V = 514, p = 0.013), with no significant change following unilateral stimulation (n = 17, p = 0.159) and a significant decrease in cortisol in the bilateral group (n = 20, V = 165, p = 0.024). Cortisone reduction was observed in the full sample (p = 0.059), with no significant change in the unilateral group (p = 0.431) and a near-significant effect in the bilateral group (p = 0.058). Changes in cortisone were moderately and positively correlated with changes in cortisol (Spearman’s ρ = 0.50, p = 0.002). Significant improvements were observed across all self-reported measures following four weeks of VNS. PHQ-9 decreased significantly – total sample n = 37, p < 0.05, unilateral p = 0.0005 and bilateral p = 0.002. GAD-7 also showed significant reductions overall (p < 0.05), and in both subgroups (p = 0.002 unilateral; p = 0.008 bilateral). PSQI improved significantly in the full sample (p < 0.05) and in both groups (unilateral p < 0.05 and bilateral p < 0.05).TVNS may reduce long-term cortisol accumulation while improving depression, anxiety, and sleep outcomes. The findings highlight bilateral stimulation as particularly effective for modulating chronic stress physiology, although further research is needed to confirm these effects.These findings support the potential of tVNS as a non-invasive intervention targeting both physiological and psychological aspects of stress-related disorders.