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Objective Alternating around-the-clock work schedules disrupt the natural circadian rhythms, resulting in circadian misalignment that increases risks for metabolic and cardiovascular diseases. How night shift work affects glycemic profiles in real-world settings remains to be settled. We hypothesized that working during the night impairs glucose levels among female hospital employees compared to shift work during the day.Material and Methods The study employs an observational design in which a subgroup of the 1001 nights-cohort in Denmark of 57 female hospital employees without known diabetes were found eligible to register working hours, sleep, and food intake in diaries while wearing a Continuous Glucose Monitor (CGM, Dexcom) for seven days. A night shift was defined as working 8 hours from 23:00 to 07:00, and a day shift as working 8 hours from 07:00 to 15:00. A ‘night off’ and a ‘day off’ were defined as not working during these intervals. The study included 39 participants, each having (1) at least one night shift and off night and/or (2) at least one day shift and off day. Thirty-five participants had both. Daily mean and standard error of glucose levels at night and day shifts and at off nights and days were calculated.Results Participants completed a total of 76 night shifts and 276 nights off, along with 72 day shifts and 246 days off. The mean glucose levels were 116.0 (SEM 1.9) mg/dL during night shifts, 113.2 (SEM 0.9) mg/dL during nights off, 111.7 (SEM 1.6) mg/dL during day shifts, and 111.7 (SEM 1.0) mg/dL during days off.Conclusion Shift work may modestly increase mean glucose levels at night but not at daytime. Results of other CGM-derived outcome variables and ongoing statistical variance component modeling using the within-person design will be presented at the conference.Funding The Danish Working Environment Research Fund (26202009; 19202209), The Health Foundation (22B0452), the European Union’s Horizon (# 874703), NFA, and The Danish Cardiovascular Academy.