PURPOSE: The study aimed to determine the differences between stress levels in day shift workers and night shift workers by measurements of hair cortisol concentration (HCC) and by using self-administered questionnaires. 
 
 METHODS: HCC was measured by using liquid chromatography-mass spectrometry. The subjective stress level was evaluated by a brief job stress questionnaire, stress response scale-18 (SRS-18), and visual analog scale (VAS).
 
 RESULTS: Mean (± standard deviation: SD) HCC in the 16 subjects was 17.28 ± 7.39 pg/mg. There was no significant difference between HCCs in day shift workers (17.98 ± 3.03 pg/mg) and rotating night shift workers (16.37 ± 1.86 pg/mg). There were also no significant differences in SRS-18 scores, job-related stress scale scores, and VAS scores between day shift workers and rotating night shift workers. There was a significant difference in HCC between the group in which the stress condition was weak or normal and the group in which the stress condition was slightly strong or definitely strong according to the SRS-18 level (p = 0.030). 
 
 CONCLUSIONS: Day shift workers and rotating night shift workers have similar HCCs and similar degrees of job-related stress. In rotating shift workers who feel strong stress, acquirement of resilience due to stress coping for medium- to long-term stress may be involved in low HCC.