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Adult Nothobranchius furzeri of the MZM-04/10 strain were individually kept and subjected to a “5+2” shifting lighting schedule (SHIFT) for 8weeks in order to evaluate the desynchronizing effects of a simulated human-like shift-work schedule on the functioning of the circadian system (CS). With this aim, sixteen 21-week-old N. furzeri were placed into a Morning, Night and Evening schedule (lights on from 08:00 to 16:00, 00:00 to 08:00 and 16:00 to 00:00h, respectively) and fed once a day in the middle of the corresponding photophase (12:00, 04:00 and 20:00h, respectively). Then, in the weekends (2days), fish were always returned to the Morning shift. As controls, 16 fish were maintained under a non-shifting LD cycle condition (CONTROL) throughout the whole experiment, with lights on from 08:00 to 16:00h.Rest-activity rhythm (RAR) of fish subjected to SHIFT showed several symptoms of chronodisruption, such as a decrease in the percentage of diurnal activity and a reduction of the relative amplitude and the circadian function index with time. When a periodogram analysis was performed, RAR of N. furzeri under SHIFT conditions showed up to three separate circadian components: one longer than 24h (26.5h) that followed the weekly 8h delays; a short-period component (˜23h) that was related to the weekend's phase advances, and finally, a 24h component.The shifting LD schedule also affected fish CS at a molecular level, with several significant differences in the expression of core genes of the molecular clock (bmal1, clock, rorα, rev-erbα) between SHIFT and CONTROL animals. RAR impairment along with changes in clock gene expression could be associated with high stress and accelerated aging in these fish.This study proposes for the first time the use of a “5+2” shifting light-dark schedule on a diurnal vertebrate model;Rest-activity rhythm of fish subjected to a shifting light-dark schedule showed several symptoms of chronodisruption;The shifting light-dark schedule affected fish circadian system at a molecular level;We found several differences in the expression of core genes of the molecular clock between control and shift animals.