This paper presents a methodological approach that can aid the assessment of variation in limnological variables in response to hydrological phases and seasonal temperature variation. The investigation was conducted in the Danube River floodplain, Kopački Rit, Croatia. The proposed approach facilitates a more accurate examination of the effects of first factor, while maintaining second factor less variable, and vice versa. Samples were collected in the floodplain channel and lake along a temporal connectivity gradient, and were grouped according to three hydrological phases (isolation, flow pulse and flood pulse) and according to three seasons (spring, summer and autumn). A permutational multivariate analysis of variance was used to analyse the hydrological effects within seasons, as well as seasonal effects within hydrological phases on selected limnological variables: pH, conductivity, dissolved oxygen, nitrites, nitrates, ammonium, total Kjeldahl nitrogen, total phosphorus, chlorophyll a concentrations and ratio of copiotrophic to oligotrophic bacteria. A multivariate data cloud was structured by hydrological phases during all investigated seasons, but not by all seasons during different hydrological phases. The strongest impact of hydrological phases on limnological variables occurred during summer. Seasonal effects were significant during the isolation and the flood pulse phases. The flow pulse phase somehow obscured the effects of seasonal change, resulting in temporal floodplain homogenization. One possible explanation is that water temperature determines potential limnological conditions, whereas hydrology determines actual conditions. Such results must be taken into account if the accurate effects of hydrology and seasonal change are to be assessed in temperate floodplain ecosystems. Copyright © 2014 John Wiley & Sons, Ltd.