Short-term streamflow regulation (hydropeaking) affects the ecology of regulated rivers. We examined the longitudinal and temporal changes occurring in fish assemblages in a hydropeaking single river reservoir between two power plants by using electrofishing along the shoreline, hydroacoustics and test fishing in the open water. A longitudinally changing fish community was found among bottom-dwelling fish in the fast-flowing and highly disturbed upstream part of the reservoir progressing to generalists and pelagic fish in the lentic and most stable environment at the downstream end. The fish assemblage showed temporal patterns as fish density increased during night-time darkness and also towards autumn. Our work provides evidence for gradient effects of flow regulation and contributes to awareness of the effects of disturbance (flow/habitat variability) on biological systems.