The present study was designed to objectively assess the effects of 3-months submarine deployment on behavioural and metabolic determinants of metabolic health. In 13 healthy, non-obese volunteers, we using stable isotope dilution, and plasma and urinary biochemistry to characterize metabolic health before and after a 3-month submarine deployment. Volunteers worked in 6-h shifts. After deployment, we observed reduced fat-free mass (mean ± SD, -4.1 ± 3.3 kg, p = 0.003) and increased adiposity (21.9 ± 3.2% fat mass to 24.4 ± 4.7%, p = 0.01). Changes in fat-free mass were positively associated with physical activity (+0.8 kg per 0.1 increase in PAL, p = 0.03). The average physical activity level was 1.64 ± 0.26 and total energy expenditure during deployment was 2937 ± 498 kcal/d, while energy intake was 3158 ± 786 kcal/d. Fasting glucose (p = 0.03), and triglycerides (p = 0.01) declined, whereas fasting free fatty acids increased (p = 0.04). Plasma vitamin D and B12 concentrations decreased (-14%, p = 0.04, and -44%, p = 0.001, respectively), and plasma calcium, and magnesium increased (+51%, p = 0.01, and +5%, p = 0.02). Haemoglobin was unchanged, but haematocrit decreased (-2.2 ± 2.1%, p = 0.005). In conclusion, submarine deployment impairs fat-free mass maintenance and promotes adiposity. High physical activity may prevent the decline in fat-free mass. Our study confirms the need to counteract Vitamin D and B12 deficiencies, and suggests impairments in erythrocyte metabolism.