<p>Fluvial ecology aims to identify patterns of river ecosystem function; however, evidence supporting its main theories derives mostly from temperate or tropical ecosystems, biasing the global extent of predictions. We studied food web subsidies along a broad range of spatial and temporal scales in a subtropical river of South America. Using C and N stable isotope analysis and standardized fish community sampling, we tested theoretical predictions of ecosystem functioning along a longitudinal gradient in a low-impact river of the Pampa biome. Bayesian mixing models for diverse trophic groups and for fish assemblage-weighted biomass showed a decrease in autochthony along a longitudinal gradient of 280&#xa0;km. This pattern remained strong and consistent across all four seasons over 2 years, independent of the irregular flood pulses. Longer food webs prevailed towards headwaters, where &gt; 90% of total fish biomass was derived from algal sources, likely due to its open canopy. Detritus was the dominant source at the lower river section, potentially due to gradual conditioning of detritus during downstream transport, increasing its nutritional quality. The observed relationship between the origin of carbon subsidies, food web length and change in food quality along the riverscape underscore the complexity of these processes. Potentially, this diversity of interconnected factors explains why none of the theoretical predictions were fully met in this ecosystem. This analysis of spatially explicit food web subsidies along river gradients contributes to understanding the mechanisms driving variability in carbon fluxes at regional scales and moving towards a more integrated global fluvial theory.</p>