Background: Working for extended hours in a physically and mentally demanding profession has subjected nurses to occupational fatigue. Limited evidence exists about nurse fatigue and alertness changes throughout shift work and their relationship with medication errors and near misses. Purpose: The purposes of this study were to: (1) assess the relationship between nurses’ fatigue and alertness, (2) evaluate nurses’ fatigue and alertness changes throughout their shift, and (3) examine the relationship between nurses’ fatigue, alertness, and medication errors and near misses. Methods: This prospective study is part of a larger mixed-method study. Fatigue and alertness data from 14 work and non-workdays were collected from a convenience sample of 90 nurses. A wearable actigraph (ReadibandTM) was used to measure alertness, while ecological momentary assessment (EMA) using text messaging was used to measure nurses’ fatigue. Results: A 1-unit increase in fatigue was associated with a 1.06-unit reduction in nurses’ alertness score (β = –1.06, 95% CI: [–1.33, –0.78], p < .01). Night-shift nurses experienced a 31-point reduction in alertness from the start to the end of the work shift. Nurses’ fatigue, but not alertness, was associated with medication errors and near misses (OR = 1.26, 95% CI [1.07, 1.48], p = .01). Conclusion: Initiating fatigue mitigation measures during mid-shift, especially for night-shift nurses, may be a viable option to mitigate fatigue and alertness deterioration among nurses and to maintain patient safety. The multifaceted nature of fatigue, as captured by EMA, is a stronger predictor of medication errors and near misses than device-measured alertness.