The aim of this laboratory study was to investigate acute effects of experimental muscle pain on spatial electromyographic (EMG) activity of the trapezius muscle during computer work with active and passive pauses. Twelve healthy male subjects performed four sessions of computer work for 2 min in one day, with passive (relax) and active (30% maximum voluntary contraction of shoulder elevation) pauses given every 40 s without and with presence of experimental pain. Surface EMG signals were recorded from four parts of the trapezius. The centroid of exposure variation analysis along the time axis was lower during computer work with active pauses when compared with passive one in all muscle parts (P < 0.05). In presence of experimental pain, EMG amplitude increased in transverse and ascending parts and relative rest time decreased in ascending part. The results of this study showed a more variable trapezius activity pattern and increased activity with active compared with passive pauses, a lowered trapezius rest with presence of experimental pain, and increased activity in the transverse and ascending parts of trapezius due to experimental pain during computer work. Acute pain led to muscle activation pattern during computer work considered to increase the risk of developing work-related musculoskeletal disorders.