This study aimed to analyze individual cortisol levels in relation to work conditions, sleep, and health parameters among truck drivers working day shifts (n = 21) compared to those working irregular shifts (n = 21). A total of 42 male truck drivers (39.8 ± 6.2 yrs) completed questionnaires about sociodemographics, job content, work environment, health, and lifestyle. Rest-activity profiles were measured using actigraphy, and cardiovascular blood parameters were collected. Salivary cortisol samples were obtained (i) at waking time, (ii) 30 min after waking, and (iii) at bedtime, during both one workday and one off day from work. Irregular shift workers showed significantly higher waist-hip ratio, VLDL-cholesterol, tiredness after work, years working as a driver, truck vibration, and less job demand compared to day workers (p < .05). High cortisol levels in irregular shiftworkers were correlated with certain stressors, such as short sleep duration and low job satisfaction, and to metabolic parameters, such as total cholesterol, HDL, LDL, VLDL, and triglycerides. Day workers had higher cortisol levels collected 30 min after waking (p = .03) and a higher cortisol awakening response (CAR, p = .02) during workdays compared to off days . Irregular shiftworkers had higher cortisol levels on their off days compared to day shiftworkers (p = .03). In conclusion, for the day workers, a higher cortisol response was observed on workdays compared to off days. Although no direct comparisons could be made between groups for work days, on off days the irregular shiftworkers had higher cortisol levels compared to day workers, suggesting a prolonged stress response in the irregular group.In addition, cortisol levels were correlated to stressors and metabolic parameters. Future studies are warranted to investigate further stress responses in the context of irregular work hours. (Author's correspondence: crmoreno@usp.br)