The daily fluctuating discharge from hydroelectric power plants, known as hydropeaking, has been shown to cause catastrophic drift in aquatic insect communities and limit secondary production, but relatively little attention has been given to its effects on periphyton, an important food resource for consumers. We simulated daily 5-h hydropeaking events over the course of 5 days in spring and summer in an open air, experimental flume system fed by a pristine 2nd order stream in the Italian Alps. We hypothesized that hydropeaking would suppress periphyton biomass and especially nutritional quality (i.e., fatty acid content). Hydropeaking resulted in decreased periphyton Chl-a and AFDM on tiles, but there was no corresponding loss on wood. Hydropeaking did not alter periphyton elemental nutrient stoichiometry but led to a disproportionate loss of periphyton fatty acid content on both substrates. Ordination of overall fatty acid profiles indicated different periphyton fatty acid profiles by substrate and a shift from physiologically important highly-unsaturated fatty acids to non-essential saturated fatty acids after hydropeaking. These results suggest that hydropeaking may have the potential to depress primary biomass and nutritional quality in downstream ecosystems, and that availability of wood substrate may mitigate part, but not all, of this effect. Since food nutritional quality, especially fatty acid content, has been suggested to be a limiting resource on production in aquatic systems, this may generate an indirect and potentially overlooked limiting effect on aquatic consumers in hydropeaking-impacted alpine rivers.