A multi-integrative approach was used to identify spatial and temporal relationships of natural and anthropogenic environmental variables affecting riverine ecosystem structure and function in the Athabasca Oil Sands Region (AOSR). A series of inter-related field studies were conducted to assess three key components of the freshwater food web (physico-chemical environment, basal productivity, benthic macroinvertebrates) utilizing an a priori environmental disturbance gradient experimental design. The gradient design was formulated to best discriminate the possible effects of natural and anthropogenic environmental variables on two river basins (Steepbank and Ells Rivers) each having different levels of oil sands (OS) land use disturbance. Findings from this study showed that natural variation explained most longitudinal and seasonal responses of physico-chemical environmental variables for both rivers, including possible mechanisms such as physical and chemical effects from the OS geological deposit and inputs from shallow groundwater upwelling. Basal productivity was likely controlled by natural variables within the Steepbank and Ells Rivers, such as potential OS deposit effects, nutrient availability and influences from turbidity and physical factors, with disturbance from OS development either negligible or not detected. Longitudinal and seasonal differences in benthic macroinvertebrate community composition were mostly related to natural variation, including possible mechanisms such as high discharge and sediment slump events on the Steepbank River, and community shifts from elevated metal concentrations from natural sources at upstream sites on the Ells River. This study demonstrated that developing baseline information on watersheds can be essential at discriminating sources of disturbance, with natural variation potentially confounding with anthropogenic factors. This study also highlights the need for further research to obtain an improved understanding of mechanistic pathways to better determine natural and anthropogenic non-point source disturbances and cumulative effects on the structure and function of tributary ecosystems in the AOSR at relevant spatial and temporal scales.