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Objective: The aim of this study was to investigate whether there is an association between hair cortisol concentrations and acute stress symptoms in family members of critically ill patients., Methods: A cross-sectional study was conducted in an adult intensive care unit of a tertiary hospital in Porto Alegre, Brazil, from August 2021 to February 2022. Family members of intensive care unit patients admitted for more than 10 days were approached for enrollment. We collected sociodemographic data and assessed resilience, religiosity, and symptoms of acute stress among family members. Samples of family members' hair were collected shortly after the interview to measure the hair cortisol concentration., Results: A total of 110 family members were included in this study. Eighty-eight (80.0%) family members presented with symptoms of acute stress. The median hair cortisol concentration was 2.37pg/mg (1.16 - 5.06pg/mg). There was no significant difference in hair cortisol concentration between family members with and without acute stress symptoms (p = 0.419). According to the multivariate analysis, only the fact that the patient was alert at the time of the family member's interview was significantly associated with the prevalence of acute stress symptoms in the family member., Conclusion: We did not find an association between the hair cortisol concentration of family members in hair segments in the months prior to admission to the intensive care unit and the occurrence of acute stress symptoms.