Abstract Introduction In shift workers, short sleep duration combined with circadian misalignment may affect behaviors that impact regulation of energy balance and metabolism. We conducted a combined field-and-laboratory study to determine how real-life shift work affects diet, physical activity, and sleep via objective and self-report measures. Methods Participants were day (n=12) and night (n=12) shift workers from an urban hospital setting (nurses and technicians, all female). During the field portion of the study, participants wore a wrist-mounted accelerometer to track sleep and physical activity during their series of shifts, and completed a computer-assisted 24-hour dietary recall. After awakening from the sleep episode following the final work shift, participants entered the laboratory in the fasted state and underwent an ad libitum 14-item test-meal buffet to objectively quantify food choice and intake. Results Sleep duration was significantly shorter and worse quality in night vs. day workers. Physical activity levels were not different between groups. Based on 24-h dietary recall, night vs. day workers consumed less protein (65.9 ± 39.0 vs. 87.2 ± 40.7 g, p=0.01) and fiber (12.5 ± 6.0 vs. 16.9 ± 6.2 g, p=0.01), but did not differ in daily intakes of calories, fat, or carbohydrate. Night vs. day workers reported a longer daily window of eating duration (14.2 ± 3.8 vs. 12.0 ± 1.5 h, p=0.02). In the lab test-meal, there were no group differences in total calories consumed. When expressed as percent of calories consumed, night vs. day workers had lower protein intake (11.82 ± 4.05 vs. 16.03 ± 5.69 %; p=0.05). Conclusion To our knowledge, this was the first study to include a laboratory-based behavioral assessment of food choice/intake in real-life night and day shift workers using objective measures. We did not assess measures of circadian phase so can only assume that circadian misalignment, in addition to the disturbances in sleep duration and quality, contributes to findings. Changes to dietary patterns in night vs. day workers (namely, reduced protein intake which may affect satiety, and prolonged daily eating duration window) may present potential pathways by which night shift work contributes to risk for overweight and obesity. Support UL1TR000040