Human alteration of large rivers is commonplace, often resulting in significant changes in flow characteristics. We used a time series approach to examine daily mean flow data from locations throughout the mainstem Missouri River. Data from a pre-alteration period (1925–1948) were compared with a post-alteration period (1967–1996), with separate analyses conducted using either data from the entire year or restricted to the spring fish spawning period (1 April–30 June). Daily mean flows were significantly higher during the post-alteration period at all locations. Flow variability was markedly reduced during the post-alteration period as a probable result of flow regulation and climatological shifts. Daily mean flow during the spring fish spawning period was significantly lower during the post-alteration period at the most highly altered locations in the middle portion of the river, but unchanged at the least altered locations in the upper and lower port ions of the river. Our data also corroborate other analyses, using alternate statistical approaches, that suggest similar changes to the Missouri River system. Our results suggest human alterations on the Missouri River, particularly in the middle portion most strongly affected by impoundments and channelization, have resulted in changes to the natural flow regime.