Fatigue-related impairments in the nursing workforce contribute to a multitude of health, safety, and economic consequences at the individual, organizational and societal levels. Long and compressed work schedules are commonly worked in the healthcare industry, but more research is needed to understand the cumulative effects of multiple work shifts on physiology-based performance outcomes in nurses. The purpose of this study was to compare the effects of a single nursing work shift versus three compressed (one every 24 hours) 12 hour shifts on performance-based fatigue in nurses and aides. Twenty-six fulltime hospital working nurses and aides (age = 36.1 ± 13.3 years) reported to the lab for testing before, immediately after working a single 12 hour shift, and after working three 12 hour shifts in a 72 hour period. Outcome measures included vigilance-based reaction time, lapses of attention, and muscle function assessments (lower and upper body muscle strength, explosive strength and vertical jump performance). All variables except hand grip strength showed a significant decline following the three work shifts. The psychomotor vigilance reaction time and lapses of attention variables also generally showed a significant decline from the end of shift one to the end of shift three, indicting an accumulation of fatigue in these metrics with increasing number of shifts worked. Muscle function variables responded early in the duty cycle, showing a significant decline after a single work shift, but did no further decline by the end of the third shift. These findings use objective measures to substantiate that fatigue impairments occur from working a single 12 hour shift, and in several instances, increase further with more successive work shifts. Caution should be employed by personnel and administrators with work schedules involving multiple compressed 12 hour shifts. Fatigue management strategies may be used to improve risks and consequences from fatigue-related mishaps, and this study reports several variables that appear sensitive to identifying and tracking fatigue in this population.