Burnout syndrome has been reported among health workers, particularly those working in critical areas, and is considered a significant public health problem. This study aimed to investigate the relationship between chronotype and work-related stress, as measured by salivary cortisol levels and burnout, among health professionals working in neonatal intensive care units. A cross-sectional study was conducted across four public hospitals in Fortaleza, Ceará, Brazil. Two hundred and fifty-six health professionals were administered the brazilian version of the Burnout Characterization Scale, the morningness–eveningness questionnaire, for chronotype, a sociodemographic questionnaire that included lifestyle habits and a salivary cortisol test. The results indicated that morning chronotype workers were significantly associated with the following: advanced age (p< 0.001), female gender (p= 0.032), married status (p= 0.014), and having children (p= 0.030) compared to those with evening and intermediate chronotypes. However, no significant association was found between signs of burnout syndrome and chronotype (p= 0.316). Participants whose work shift did not match their chronotype had significantly higher initial salivary cortisol levels (p= 0.013). The findings suggest that adapting working hours to an individual’s biological rhythm can help mitigate potential negative effects on physical and mental health. Thus, it is recommended that professionals’ working hours be adjusted accordingly.