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Background and purpose: Efforts to improve nurses' physical and mental health are critical to ensuring the safety and quality of the healthcare system. Long-term studies targeting the relevancy of nurses' occupation characteristics with health conditions remain insufficient. This study aimed to examine the relationship between nurses' night shift and sleep problems and metabolic abnormalities risk. Methods: This study was a part of the National Nurse Health Study, an ambispective cohort study in China, in 2021. Based on an integration physical examination data system, this study carried out a retrospective analysis of 730 nurses from 2018 to 2020 and combined with a questionnaire survey in 2021. The STROBE guidelines were adopted for reporting. Results: In the 23 (23.0, 24.0) months follow-up, higher night shift load was associated with more sleep problems such as shortened sleep duration, sleep disorders, poor sleep quality, and sleep deprivation. Moreover, night shift load was associated with chronic diseases risk factors, increasing body mass index and body fat, with more night shift density, increasing the occurrence of low levels of high-density lipoprotein cholesterol, high triglyceride, triglyceride/high-density lipoprotein cholesterol ratio, and serum uric acid. Conclusion: The night shift load has become an occupational health concern, contributing to chronic diseases relevant metabolic risk factors and negative influence on sleep health. Focus on the strategies to improve the sleep quality of nurses undergoing night shift work, optimize work scheduling and ongoing monitor the relevant risk factors are essential to enhance the stability and well-being of the nursing workforce. Clinical Trials registration information: NCT04572347, on October 1, 2020. https://www.clinicaltrials.gov/ct2/show/NCT04572347 [ABSTRACT FROM AUTHOR]