Recirculating aquaculture systems (RAS), offering many economic and fish husbandry benefits, are characterized by an accumulation of dissolved organic matter (DOM) and, specifically, humic substances (HS). As reported in a number of studies, HS may affect biological activity in both invertebrates and vertebrates. Given the accumulation of HS in RAS, it is therefore of great interest to characterize DOM and, specifically, its HS fraction in the RAS. The present study was aimed at characterizing long-term changes in fluorescent DOM composition in the culture water of RAS systems, which were operated in a novel, zero water exchange mode. Two such zero-discharge recirculating systems (ZDS) were examined: a freshwater system, stocked with hybrid tilapia (Oreochromis aureus x Oreochromis niloticus) and a marine system, stocked with gilthead seabream (Sparus aurata). Excitation-emission matrices (EEMs) of fluorescence, coupled with parallel factor analysis (PARAFAC), were used to characterize and quantify the different DOM components in the ZDS. In the culture water, one tryptophan-like and four HS-like components were identified. The fluorescence intensities of three of the HS-like components as well as the tryptophan-like component increased at comparable rates during ZDS operation while a much slower accumulation of these compounds was observed in a parallel operated, flow-through, freshwater aquarium. The ZDS examined in this study comprised a sludge digestion stage where a considerable accumulation of all fluorescent components was detected. A HS-like components and a tryptophan-like component in blood of tilapia from the freshwater ZDS were similar to components found in the culture water. Blood levels of both components were higher in fish cultured in the DOM-rich ZDS than in fish raised in the control, flow-through freshwater aquarium. Fluorescence of the HS-like component found in the fish blood increased also with time of ZDS operation. The finding that fish blood contains a HS-like fluorescent component may have important implications for the understanding of the physiological effects of HS in fish and the possible benefits of these substances in aquaculture.