Retrospective and prospective studies show that stress at work is linked to an increased risk of hypertension, diabetes mellitus, and coronary heart disease. However, the nature of the contributory job stressors and biological mechanisms need further elucidation. The study is aimed to determine the associations between aspects of the occupational stress index (OSI) and arterial hypertension, diabetes mellitus (DM) type 2, and lipid disorders in working middle-aged men and women. The cross-sectional study involved 989 middle-aged men and women in different occupations. The OSI was calculated by using standardized questionnaires. The total participation rate was 93%. Occupational stressors were divided into seven groups: High Demands, Strictness, Underload, Extrinsic Time Pressure, Noxious Exposure, Avoidance, and Conflict/Uncertainty. Serum lipid levels, glucoregulation, blood pressure, and cardiovascular risk factors were measured. For both women and men, the total OSI score associated significantly with DM (women: odds ratio [OR] 2.4, 95% confidence intervals [CI] 1.67-3.45; men: 1.21, 1.15-1.45), any type of dyslipidemia (women: 1.54, 1.17-2.03; men: 1.31, 1.24-1.39), and arterial hypertension (women: 1.15, 1.10-1.21; men: 1.58, 1.49-1.68). The group as a whole showed associations between total OSI and low high-density lipoprotein (HDL) cholesterol, high total cholesterol, and high triglyceride levels. Of the OSI aspects, Underload associated significantly in both men and women with arterial hypertension (women: 3.48, 1.91-6.31; men: 2.71, 1.96-3.75) and dyslipidemia (women: 3.26, 2.13-4.99; men: 2.11, 1.76-2.52). Underload was also associated with several lipid abnormalities in the group as a whole. It associated with DM in women only (4.7, 2.84-7.81). All remaining OSI aspects also associated significantly and positively with DM in women only. Conversely, in male workers, but not female workers, High Demand, Conflict/Uncertainty, and Extrinsic Time Pressure associated significantly with arterial hypertension. Strictness and Conflict/Uncertainty associated positively with dyslipidemia in women only. Noxious Exposures associated positively with DM and arterial hypertension in women only. The study provides evidence for the association of work stress with metabolic disorders and hypertension. Total OSI associated significantly with DM type 2, arterial hypertension, and dyslipidemia in both genders. Different OSI aspects associated with these health issues in gender- and occupational-specific patterns. Underload, which represents lack of social communication, simple task preparation, and underestimation of working results, associated most strongly of all OSI aspects with disease in both the sexes.