Nurses report that they are required to work during their scheduled breaks and generally experience extended work times and heavy workloads due to staffing shortages. This study aimed to examine changes in personal, work-related, and overall stress, as well as biological responses and fatigue experienced by nurses during three consecutive 12 h workdays (i.e., the typical “three-twelves” schedule). We also considered the moderating effects of social resources. This prospective study of 81 medical/surgical nurses who completed questionnaires and provided saliva samples at four designated intervals (i.e., pre-shift and post-shift on workdays 1 and 3). Fatigue reported by night shift nurses increased significantly over three consecutive workdays (p = 0.001). Day shift nurses said they encountered more social support than those on the night shift (p = 0.05). Social support moderated the relationship between work-related stress at baseline and reported fatigue on day 3.