Biomarker assays are widely used as proxies for contaminant-induced effects in aquatic organisms. However, in many cases, their intrinsic natural variability due to exogenous and endogenous factors makes the interpretation of biomarker data difficult. In the present study, we investigated the natural fluctuations of six hepatic biomarkers, namely ethoxyresorufin-O-deethylase (EROD) in fish and pentoxyresorufin-O-deethylase (PROD) in crustacea, catalase (CAT), carboxylesterase (CbE), glutathione-S-transferase (GST), total glutathione peroxidase (GPX) and glutathione reductase (GR) in two deep-sea fish species, namely Alepocephalus rostratus and Lepidion lepidion and the decapod crustacean Aristeus antennatus. The NW Mediterranean deep-sea environment is characterized by very stable temperature and salinity conditions, allowing the exclusion of these two factors as potential sources of interference with biomarker activities. Biomarker results exhibited a clear influence of reproductive processes on enzyme activities, in particular in A. rostratus, which presented a pronounced seasonal pattern linked to variations in the gonadosomatic index (GSI). In addition, other factors such as food availability may also have influenced the observed variability, in particular in specimens of L. lepidion, which did not exhibit variations in reproductive activity throughout the sampling period. Depth-related variability did not exhibit a clear trend and fluctuations across sampling depths were not attributable to any specific factor. Body size had also a significant influence on some biomarkers, although allometric scaling of certain enzyme activities appears to be species-specific. The present work has thus shown that despite the lack of fluctuations of abiotic parameters such as temperature and salinity, biomarker activities in deep-sea organisms still exhibit significant variability, mainly as a result of reproductive processes and food availability.