Background: Medical students often experience high levels of stress due to adverse study conditions, which may have adverse health consequences. Hair cortisol concentration (HCC) has been described as a physiological marker for chronic stress and might thus help to identify students under stress and examine the study conditions being responsible for long-term physiological stress responses. This study therefore investigated the association between study conditions and HCC in a sample of medical students. Methods: Fifty-five students from a medical school in Germany completed a paper-based questionnaire and had hair samples collected between July 2020 and July 2021. Study conditions were assessed with student versions of questionnaires based on the Job-Demand-Control-Support model (StrukStud, 25 items) and Effort-Reward Imbalance model (Student ERI, nine items). HCC of two centimeters closest to the scalp were determined by a cortisol luminescence immunoassay. Linear multiple regression analyses were performed to examine associations between study conditions and HCC. Results: Demands (B = 0.23, p = 0.002), effort (B = 0.12, p = 0.029) and the effort-reward-ratio (B = 0.28, p = 0.007) were positively associated with HCC in separate regression analyses, adjusted for age and sex. Only the association between demands and HCC remained significant when all components of the respective questionnaire were considered in the same model (B = 0.22, p = 0.003). Conclusion: The results suggest that adverse study conditions may be associated with activation of the hypothalamic-pituitary-adrenal axis stress response as reflected by increased HCC. Longitudinal research is needed to confirm these cross-sectional results and examine effects of more prolonged stress due to adverse study conditions.