Running waters are among the most threatened ecosystems globally, having altered hydrological regimes, homogenized habitat, and impaired water quality. These multiple stressors impact aquatic biodiversity and ecosystem function across space and time, although a clear mechanistic understanding is still lacking. Here, we examined the trophic response of macroinvertebrates among streams in a Swiss lowland catchment encompassing a gradient of land uses. Clear compositional changes were observed as anthropogenic impacts increased from least-impacted to agricultural and urbanized sites. Taxonomic diversity was lowest at sites with morphological and water quality impairment (agricultural sites), whereas taxonomic identity (susceptible vs. generalist species) mainly changed due to water quality degradation (agricultural and urban sites) based on the SPEAR (pesticides) index. Using stable isotopes (δ13C, δ15N), a simplification in macroinvertebrate trophic structure was evident along the land use gradient. At a site receiving wastewater treatment effluent, stable isotopes also revealed trophic shifts in primary consumers that corresponded to changes in available food resources. Results further showed that some taxa losses, e.g., the mayfly Ecdyonurus, to land- use effects may be due to low trophic plasticity. The combination of analyses, including stable isotopes, provided an improved mechanistic understanding of community and population responses to land-use changes along river networks.