OBJECTIVES: To investigate the physiological effects of rotational shift work on measures of cardio-metabolic function. METHODS: Sedentary, healthy men (n = 87; age 37 +/- 9 years; body mass index: 30.7 +/- 5.1 kg m2) were recruited and categorized via occupation. SHIFT group: currently employed in rotational shift work defined by 8-12 h morning, afternoon, and night rotations; or NSHIFT: working fixed daytime hours. Testing procedures included baseline objective sleep assessment and laboratory testing, conducted between 0600 and 0900 h to assess body composition, cardiorespiratory fitness (VO2peak), inflammatory status [C-reactive protein, interleukin (IL)-6, and tumour necrosis factor-alpha (TNF-alpha)], glucose metabolism, heart rate variability (HRV), and self-reported leisure time physical activity (PA). RESULTS: SHIFT reported significantly less leisure time PA (P = 0.019), reduced VO2peak (P = 0.007), higher body fat percentage (BF%) (P = 0.021), increase response time to oral glucose tolerance test (P = 0.016), and higher IL-6 values (P = 0.008) compared with NSHIFT. A significant difference was observed in actigraphy measured total sleep time, with SHIFT recording reduced sleep following a night shift (P = 0.001). No group difference was observed in HRV or average sleep parameters (P > 0.05). Linear regression identified a significant association between occupation and inflammatory status (P = 0.006). CONCLUSIONS: Rotational shift work is associated with increased risk factors for cardio-metabolic disorders, despite no differences in sleep quality and quantity. The results suggest rotational shift work has a detrimental effect on the health and wellbeing of employees; with homeostatic desynchronization identified as potential pathogenic mechanisms.