Flow is widely considered one of the primary drivers of instream ecological response. Increasingly, hydroecological models form the basis of integrated and sustainable approaches to river management, linking flow to ecological response. In doing so, the most ecologically relevant hydrological variables should be selected. Some studies have observed a delayed macro-invertebrate (ecological) response to these variables (i.e. a cumulative inter-annual effect, referred to as multi-annual) in groundwater-fed rivers. To date, only limited research has been performed investigating this phenomenon. This paper examines the ecological response to multi-annual flow indicators for a groundwater-fed river. Relationships between instream ecology and flow were investigated by means of a novel methodological framework developed by integrating statistical data analysis and modelling techniques, such as principal component analysis and multistep regression approaches. Results demonstrated a strong multi-annual multi-seasonal effect. Inclusion of additional antecedent flows indicators appears to enhance overall model performance (in some cases, goodness of fit statistics such as the adjusted R-squared value exceeded 0.6). These results strongly suggest that, in order to understand potential changes to instream ecology arising from changing flow regimes, multi-annual and multi-seasonal relationships should be considered in hydroecological modelling.