AbstractRiver scientists strive to understand how streamflow regimes vary across space and time because it is fundamental to predicting the impacts of climate change and human activities on riverine ecosystems. Here we tested whether flow periodicity differs between rivers that are regulated or unregulated by large dams, and whether dominant periodicities change over time in response to dam regulation. These questions were addressed by calculating wavelet power at different timescales, ranging from 6 hr to 10 years, across 175 pairs of dam‐regulated and unregulated USGS gages with long‐term discharge data, spanning the conterminous United States. We then focused on eight focal reservoirs with high‐quality and high‐frequency data to examine the spectral signatures of dam‐induced flow alteration and their time‐varying nature. We found that regulation by dams induces changes not only in flow magnitude and variability, but also in the dominant periodicities of a river's flow regime. Our analysis also revealed that dams generally alter multi‐annual and annual periodicity to a higher extent than seasonal or daily periodicity. Based on the focal reservoirs, we illustrate that alteration of flow periodicity is time varying, with dam operations (e.g., daily peaking vs. baseload operation), changes in dam capacity, and environmental policies shifting the relative importance of periodicities over time. Our analysis demonstrates the pervasiveness of human signatures now characterizing the U.S. rivers' flow regimes, and may inform the restoration of environmental periodicity downstream of reservoirs via controlled flow releases—a critical need in light of new damming and dam retrofitting for hydropower globally.