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Objective The International Agency for Research on Cancer (IARC) recently classified night work as a probable human carcinogen and highlighted the lack of mechanistic studies in real-world settings. This study investigates behavioral and biological pathways linking night work to cancer and obesity risk, and explores the role of sleep and circadian disruption.Methods As part of the SHIFT2HEALTH project, a mechanistic study is being conducted recruiting 200 night shift and 200 day workers from multiple occupational sectors in Austria and The Netherlands. All participants complete questionnaires on work schedules, lifestyle, diet, and sleep. Participants provide fasting blood samples (for inflammatory, oxidative stress, and appetite hormone markers), first-morning urine (for melatonin), stool samples (for gut microbiome), hair samples (for chronotype). Night shift workers additionally collect 24-hour urine samples across a night and a day shift (for 6-sulfatoxymelatonin rhythm), dried blood spots pre- and post-shifts (for metabolomics), repeated tongue swabs (for oral microbiome) and wear and actiwatch for 7–10 days capturing sleep, activity, and light exposure continuously. We will use linear regression models with biomarker levels as the outcomes and night work as the exposure adjusting for potential confounders.Expected Results We will present prior mechanistic evidence, the study protocol and preliminary results from the SHIFT2HEALTH project on a variety of biomarker levels including hormones and inflammatory markers linked to night work. We will also explore the role of sleep and circadian misalignment based on actigraphy data, and assess susceptibility to night shift work according to sleep characteristics and chronotype.Conclusion The SHIFT2HEALTH study is among the most comprehensive real-world mechanistic studies of night shift work to date. The results will help identify key biological pathways underlying increased carcinogenic and metabolic risks, supporting future risk stratification and targeted occupational health interventions.Funding Research related to this abstract was funded by the EU HEALTH call ‘HORIZON-HLTH-2022-STAYHLTH-01-05 (Grant agreement ID: 101080788)