The present study aimed to evaluate the effect of sex isolation on the off-season gamete quality in pikeperch (Sander lucioperca) upon the transport of fish from outdoor to indoor conditions and the shift of their cycle for six months in comparison to natural. In total 128 three-year-old fish (1.4 ± 0.3 kg) were transferred from an in-pond raceway to recirculation aquaculture system and submitted to one-year-long examination. At the time of transport, the fish were in the stage of late vitelogenesis/spermatogenesis. Three tanks were stocked with either males only (ISO males), females only (ISO females), or combined both sexes within each tank (MIX). Further on, fish were submitted to fully controlled photo-thermal conditions, composed of a period of constant temperature and photoperiod (resorption), followed by stable temperature and decreasing photoperiod (regeneration), continued photoperiod reduction with slow cooling (vitellogenesis) and finished with constant low temperature and increasing photoperiod (maturation). Finally, once becoming ready for hormonal stimulation, fish were warmed to a spawning temperature of 12 °C, and ovulation induction took place. Next to the main parameters of gamete quality, the physiology of the sex steroids and histopathology of gonads, respectively, were monitored over the entire course of the study. Both males and females from the MIX group showed better gamete quality, best witnessed in significantly higher embryo survival (71.4 ± 10.8 % vs. 46.0 ± 26.8 %) and sperm curvilinear velocity (120.3 ± 11.8 μm/s vs. 94.6 ± 20.4 μm/s). Although characterized with a higher final maturation stage at the time of injection, latency time in ISO females was shorter. Both histological and physiological examinations showed undisturbed gonadogenesis in both genders upon reproductive cycle shift. The paper suggests broodstock maintenance in a mixed-gender manner and describes solutions for utilizing novel method for high gamete quality and reduced energy costs.