The association of fast backward-rotating shift work (ie, anticlockwise sequence of afternoon, morning, and night shifts) with subjective and objective measures of sleep-wake quality, daytime attention, and tiredness of health care workers has not yet been established. To investigate the association of shift rotation direction with tiredness, sleepiness, and sustained attention among nurses working forward- and backward-rotating shifts. Data of this cohort study were collected from nurses working at 5 midsized Italian hospitals. The nurses had either a forward-rotating schedule (ie, morning to afternoon to night) and or a backward-rotating schedule (ie, afternoon to morning to night). The data were collected from July 2017 to February 2020. Data analysis was performed from May to October 2020. Participants were working either forward- or backward-rotating schedules, in which the sequence of 3 shifts (morning, afternoon, and night) changed in a clockwise or anticlockwise direction. Sleep data were collected using the Karolinska Sleepiness Scale and Pittsburgh Sleep Quality Index. Sustained attention was measured using the Psychomotor Vigilance Task. Tiredness was evaluated using the Tiredness Symptom Scale. A total of 144 nurses (mean [SE] age, 41.3 [0.8] years; 92 women [63.9%]) participated in the study; 80 nurses had forward-rotating schedules, and 64 had backward-rotating schedules. Nurses with irregular sleep-wake patterns due to night shift work had poor sleep quality (46 [57.5%] in forward-rotating schedule group; 37 [57.8%] in backward-rotating schedule group). Nurses working backward-rotating shifts exhibited significantly greater sleepiness (F1,139 = 41.23; P < .001) and cognitive slowing (ie, longer median reaction times; F1,139 = 42.12; P < .001) than those working forward rotations. Importantly, these differences were not affected by age, years of employment, and quality of sleep. Of nurses working on a backward-rotating schedule, 60 (93.8%) reported elevated sleepiness (Karolinska Sleepiness Scale score ≥7) after the night shift. The median reaction time (F1,139 = 42.12; P < .001), 10% fastest reaction time (F1,139 = 97.07; P < .001), minor lapses (F1,139 = 46.29; P < .001), and reaction time distribution (F1,139 = 60.13; P < .001) of nurses on backward-rotating schedules indicated a lower level of vigilance, which is negatively associated with neurobehavioral performance. In this study, both shift rotation models were negatively associated with health and cognitive performance. These findings suggest that forward shift rotation may be more beneficial than backward rotation for several measured performance attentional outcomes and sleepiness. Optimization of shift rotations should be implemented to decrease the combination of the negative outcomes associated with shift work and reduce the potential risk of medical errors in health care systems.