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A healthy state of life suggests not only a disease‐free condition but also normal psychological functioning and behaviour. To maintain a healthy life, the duration of light exposure is a crucial factor. Perturbation of the standard light–dark cycle (LD: 12 h light‐12 h dark in mice) may result in brain, behavioural and physiological abnormalities. The current study determined the effects of 3 and 5 weeks of constant darkness (DD: 00 h light‐24 h dark) on the behaviour, hormones, prefrontal cortex (PFC) and metabolome of male and female C57BL/6 J mice. We also studied 3 weeks of restoration in LD following 5 weeks of DD exposure. The results revealed that 3 weeks of DD affected male mice more than females, and 5 weeks of DD had a comparable impact on behaviour, hormones and the PFC of male and female mice. After restoration in LD, the DD‐induced changes reverted to time‐matched LD conditions in male and female mice. Furthermore, metabolome analysis corroborated male and female mice's behavioural and molecular kinetics. The present study laid the foundation for understanding how DD affects behaviour and the PFC as a function of (a) time and (b) sex and described the roles of stress and sex hormones, cytokines, neurotrophins and metabolic pathways. [ABSTRACT FROM AUTHOR]