This study identified the nursing work activities that could be the primary sources of work-related acute fatigue in US hospital nurses. Continuous recording of working heart rate and random observations of nursing activities were applied to collect data from eight nurses during two consecutive 12 h day shifts. Using descriptive statistics and random-effect analysis of variance, the contributions of individual nursing work activities to acute fatigue were compared based on the activity frequencies and nurses' corresponding heart rate elevations. Of 860 observed nursing-related work activities, manual patient-handling, bedside-care, care-coordinating, and walking/standing activities accounted for 5%, 16%, 38%, and 41%, respectively. After controlling for the differences of participant and shift, the percentage of working heart rate to maximal heart rate of manual patient-handling (64.3%), bedside-care (59.7%), and walking/standing (57.4%) activities were significantly higher than that of care-coordinating activities (52.3%, F[3, 38.0]  = 7.5, P < 0.001). These findings suggest that bedside care and walking/standing, other than manual patient handling, contributed most to the level of acute fatigue.