Objective :Shift work has been linked to cardio-metabolic diseases, but insight into different shift work-related aspects and chronotype of shift workers and their relation with metabolic risk factors is limited. This study examined the association between current shift work status, frequency and duration of night shift work, chronotype, and metabolic risk factors in a population of health care workers. Methods: Anthropometrics, questionnaires, and blood samples were collected from 503 shift working and 93 non-shift working health care workers employed in hospitals. Body mass index, waist circumference, cholesterol (total, HDL, LDL), triglycerides, and high-sensitivity C-reactive protein were measured. Associations of current shift work, frequency (non-night shift worker, 1-2, 3-4, ≥5 night shifts/month) and duration of night shift work (non-night shift workers, <10, 10-19, ≥20 years), and shift workers' chronotype, with metabolic risk factors were studied using linear regression analysis. Results: Compared to non-shift workers, shift workers' total cholesterol level was 0.38 mmol/L lower (95%-CI = -0.73 -0.04) and LDL cholesterol was 0.34 mmol/L lower (95%-CI = -0.60 -0.08). For all other metabolic risk factors, no differences were found. The association between shift work and LDL cholesterol was especially found among shift workers working night shifts for ≥20 years (B = -0.49 (95%-CI = -0.78 -0.19)). No differences were found for night shift frequency and chronotype. Conclusion: In this population of health care workers employed in hospitals, no evidence for differences in metabolic risk factors was observed that could underlie a link between shift work and car-dio-metabolic diseases. Further research using different aspects of shift work to study the association with metabolic risk factors is recommended.