AbstractLong‐term monitoring of the food web of a regulated hydropeaking river was conducted to assess if previously documented effects of changing ramping rates (RRs) were maintained with the addition of 6 years of data. Using carbon and nitrogen stable isotope analyses, we hypothesized that: (1) macroinvertebrates and fish inhabiting areas below peaking hydrodams would be higher in δ15N and lower in δ13C due to increased flow velocity and the influence of light respired dissolved inorganic carbon, relative to those sampled from areas with a natural flow regime; (2) the increase in δ15N of macroinvertebrates would lead a shorter food web length in the regulated river, but δ13C and niche width would be similar between the restricted and unrestricted RR periods (i.e., the BACI analysis); and (3) isotopic metrics (e.g., δ13C, δ15N, niche width [SEAB], and food chain length [Δ15N]) would correlate with variations in flow characteristics through time. Consistent with previous analysis conducted over a shorter time period, a shift toward higher δ15N values was observed for both fish and invertebrates, but, contrarily, only invertebrates (not fish) had a lower δ13C value downstream of the dam. Over the long term, the before‐after‐control‐impact analysis found no effect of RRs on any of the food web metrics, implying that the change in operation did not affect the river food web. However, analysis of the time series data indicated that flow metrics and trophic metrics were often correlated, including a negative effect of RR (invertebrates) and discharge (fish) on food chain length. This study illustrates the difficulty in detecting changes in food web structure and function under changing flow regime influenced by natural and anthropogenic effects. As such, this study highlights the need for considering large spatial and temporal scales to differentiate between confounding effects of climate, natural variability, and altered flow regimes on food webs in regulated rivers.