Stress is the most frequent seizure-precipitating factor reported by patients with epilepsy, while stressful life events may increase seizure susceptibility in humans. In this study, we investigated the relations between both biological and behavioral measures of stress in children with a first epileptic seizure (hereafter called seizure). We hypothesized that hair cortisol, a biomarker of chronic stress reflecting approximately 3months of preceding exposure, might be increased in children with a first seizure. We also employed standardized questionnaires to examine presence of stress-related behavioral markers. This was a cross-sectional clinical study investigating stress-related parameters in children with a first seizure (First Epileptic Seizure Group (FESG), n=22) in comparison to healthy children without seizures (Control Group, n=29). Within 24h after a first seizure, hair samples were collected from children for the determination of cortisol. In parallel, perceived stress and anxiety and depressive symptoms were examined with appropriate self- and parent-completed questionnaires, and history of stressful life events during the past year was recorded. Emotional and behavioral problems were also assessed by parent-reported validated and widely-used questionnaires. Higher hair cortisol measurements were observed in the FESG than control children (7.5 versus 5.0pg/mg respectively, p=0.001). The former were more likely to complain of somatic problems than the latter (59.8 vs. 55.4 according to DSM-oriented Scale, p=0.021); however, there were no differences in perceived stress and anxiety or depressive symptoms between the two groups. Using ROC analysis of hair cortisol measurements for predicting disease status, the maximum sensitivity and specificity were observed for a cut-off point of 5.25pg/mg. Increased hair cortisol indicates chronic hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis prior to the first seizure. This might have contributed to the epileptogenesis process and may help explain the higher incidence of antecedent somatic complaints in the first seizure group.