Background: Fatigue is prevalent among hospital nurses and has been linked to medical errors and decreased patient safety. However, little is known regarding the relationship between occupational physical activity, sedentary behavior, and fatigue. Objective: To assess the impact of 12-hour shifts on nurses' fatigue and its relationship to occupational physical activity and sedentary behavior. Design: Prospective-cohort study design Setting(s): Midwestern trauma one academic medical center Participants: A total of 80 registered nurses working 12-hour day and night shifts participated in this study and completed momentary measures of fatigue (texting, aim one). Only 52 participants were included in aim two analyses (included activity monitoring, aim two). Methods: Occupational patterns of momentary fatigue was measured via ecological momentary assessments. Occupational physical activity and sedentary behaviors (e.g., step count, time spent sitting, standing, and walking) were measured for 14 continuous days using the ActivPAL3 micro activity monitor. Mixed models were used to examine the effects of shift type and time within a shift on occupational fatigue. General estimation equations were used to examine the relationship between time spent sitting, standing, and walking on fatigue. Results: Regardless of shift type, nurses exhibited a significant rise in fatigue; however, the rise was greater during night shifts compared to day shifts. Walking was positively associated with fatigue during day shifts, and negatively associated with fatigue during night shifts. Conclusions: The rise in fatigue was greater among nurses working night shifts compared to day shifts, which could place them at greater risk for fatigue-related consequences. The relationship between walking and fatigue was moderated by shift-type. Tweetable abstract: Nursing fatigue rises during 12-hour shifts, but the rise is greater for those working night shifts @DrRobertoBenzo.