The aim was to examine the perception of work stressors in relation to ambulatory measures of heart rate variability (HRV). Results are based on a sample of 653 healthy male workers aged 40-55 from the Belgian Physical Fitness Study conducted in 1976-1978. Data were collected by means of self-administered questionnaires and bio-clinical examinations. An index of physical and psychosocial work stressors containing five items was constructed based on the job stress questionnaire. Data on HRV were collected by means of 24-h ambulatory ECG recordings on a working day. Both time and frequency domain measures of HRV were calculated. Associations between work stressors and HRV measures were assessed by means of correlations, multiple linear regression analysis and analysis of (co)variance. The work stressor index was significantly associated with lower pNN50 (the percentage of differences between adjacent normal RR intervals > 50 ms), lower high frequency power and a higher ratio of low frequency over high frequency power. Very similar results were obtained after adjusting for age, language, occupation, smoking, body mass index, total cholesterol, systolic blood pressure and leisure time physical activity. No significant associations were found with SDNN (the standard deviation of all normal RR intervals) and low frequency power. The perception of work stressors was related to reduced parasympathetic activity in a sample of 653 healthy male workers. These findings support the idea that particularly the parasympathetic component of the autonomic nervous system is related to work stress.