In order to understand the high prevalence of musculoskeletal disorders associated with stressful work, it is important to explore the relationship between muscle activity and psychophysiological stress responses. The present real-life study examines surface trapezius electromyographic (sEMG) activity, heart rate, blood pressure, and levels of urinary catecholamines and salivary cortisol among 31 female employees working at supermarkets, where the prevalence of neck and shoulder disorders is high (60-70%). As expected, the results show that psychophysiological arousal was high during work. Significant correlations were found between self-reports indicating negative stress (stressed, exhausted, tense) and sEMG activity during work. No significant correlations were found between self-reports of positive reactions (stimulated, concentrated, happy) and sEMG activity. No associations were found between sEMG activity and pain or between negative stress ratings and pain. Objectively measured workload and physiological stress responses did not correlate significantly with sEMG activity. Thus, our data indicate that perceived negative stress may have a specific influence on muscle activity, which may be of importance for musculoskeletal disorders in jobs with low-to-moderate physical load and negative psychosocial factors.