The purpose of this study was to compare physical workload, electromyography (EMG) of the trapezius muscle, neck pain and mental well-being at work between night and day shifts in twenty Swiss nurses. Work pulse (average increase of heart rate over resting heart rate) was lower during night (27 bpm) compared to day shifts (34 bpm; p < 0.01). Relative arm acceleration also indicated less physical activity during night (82% of average) compared to day shifts (110%; p < 0.01). Rest periods were significantly longer during night shifts. Trapezius muscle rest time was longer during night (13% of shift duration) than day shifts (7%; p < 0.01) and the 50th percentile of EMG activity was smaller (p = 0.02), indicating more opportunities for muscle relaxation during night shifts. Neck pain and mental well-being at work were similar between shifts. Subjective perception of burden was similar between shifts despite less physical burden at night, suggesting there are other contributing factors.