Abstract Introduction Shift workers experience misalignment between internally-driven circadian rhythms and externally-driven behavioral schedules. Mounting evidence from hormone-, cytokine-, exosome-, proteome-, and metabolome-based biomarker analyses of blood samples predicts disruption of glucose and insulin regulatory pathways during night shift schedules. Here, we quantified circulating glucose and insulin concentrations in a constant routine (CR) protocol to assess functional metabolic outcomes of prior simulated shift work. Methods Healthy adults (N=14; aged 22–34; 4 females) completed a 7-day/6-night in-laboratory study, with randomization to either a 3-day simulated day shift (DS) schedule (n=7) with nighttime sleep (22:00–06:00) and 3 daytime meals (07:30, 13:00, 19:30), or a 3-day simulated night shift (NS) schedule (n=7) with daytime sleep (10:00–18:00) and 3 nighttime meals (19:30, 01:00, 07:30). A 24h CR protocol followed, during which participants stayed awake under constant behavioral and environmental conditions, including hourly isocaloric snacks. Serum collected via intravenous catheter every 6h during the CR was assayed using glucose colorimetric detection and human insulin ELISA. Glucose and insulin concentrations were analyzed with mixed-effects ANOVAs with fixed effects of prior simulated shift condition (day, night) and sampling time (01:30, 07:30, 13:30, 19:30) and their interaction, with a random effect over participants on the intercept. Mixed-effects cosinor regression analyses were conducted to compare endogenous circadian rhythms between conditions. Results For glucose, there was a significant condition*time interaction (F[3,30]=4.99, P=0.006), with a 9.34±2.42h delay (mean±SE) of the endogenous circadian glucose rhythm after simulated NS compared to DS (t[12]=3.85, P=0.002). For insulin, there were significant effects of condition (F[1,30]=5.93, P=0.021) and time (F[3,30]=3.01, P=0.046), with no significant endogenous rhythmicity after simulated DS, but the appearance of a circadian rhythm with reduced insulin concentrations after simulated NS (t[12]=-3.32, P=0.006). Conclusion Under constant routine, we found altered endogenous temporal patterns of glucose and insulin after just 3 days of simulated NS compared to DS. Our results indicate that biomarker and omics-based predictions of disrupted glucose and insulin regulatory pathways have functional significance evident in circulating glucose and insulin concentrations. This may explain the insulin resistance and increased risk of metabolic disorders observed in shift workers. Support (if any) Andy Hill CARE Fund FY22-POP-02 and WSU CPPS start-up funds