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In protected areas, outdoor recreation, and nature-based tourism can act as potential stressors for wildlife. Given the growing demand for nature recreation, the consequences of high tourist visitation on wildlife need to be assessed in order to avoid incompatibilities between public use and species protection goals. The Tatra National Park (Poland), in the Carpathian Mountains, is a unique alpine ecosystem visited by three million tourists per year. It hosts the only native population of an endemic subspecies of chamois (Rupicapra rupicapra tatrica). We investigated the effects of tourist disturbance, the number of visitors, and the season on the concentration of fecal cortisol metabolites (FCM) in Tatra chamois in 2009. FCM levels of chamois were significantly higher and showed higher variation at high tourist disturbance (mean ± SD, 46.2 ± 31.53 ng/g, n = 56) than at low disturbance sites (mean ± SD, 17.2 ± 8.05 ng/g, n = 38). Stress levels increased with the number of visitors and therefore showed a peak in summer, coinciding with the highest number of visitation to the national park. A large portion of chamois habitat in Tatra National Park is within the area of influence of the touristic trail network. The temporal or permanent creation of areas free of disturbance in protected areas should be considered, especially in the periods of high tourist visitation. This study highlights the need to monitor the effects of tourist activities on wildlife and to implement new policies in the management of protected areas.