Prolonged computer use, especially if fatigue ensues, is associated with visual and musculoskeletal symptoms. The aim was to determine the time-course of perceived fatigue in the wrist, forearm, shoulder and eyes during a 60-min mouse task (painting rectangles), and whether object size and/or mouse use demands were of influence. Also, we investigated performance (number of rectangles painted), and whether perceived fatigue was paralleled by local muscle fatigue or tissue oxygenation. Ten women performed the task for three conditions (crossover design). At condition 1, rectangles were 45 × 25 mm, square paint cursor size 1.3 × 1.3 mm, and mouse–pointer movement ratio 1:26. At condition 2, the same cursor size and mouse–pointer movement ratio was used, but rectangles were smaller. At condition 3, the smaller rectangles were used, but the cursor size was also smaller and mouse–pointer movement ratio was 1:8. The results showed increased self-reported fatigue over time, with the observed increase greater for the eyes, but no change in physiological responses. Condition 2 resulted in higher performance and increased eye fatigue. Perceived fatigue in the muscles or physiological responses did not differ between conditions. In conclusion, computer work tasks imposing high visual and motor demands, and with high performance, seemed to have an influence on eye fatigue.