Human-altered flow regime is often claimed to be one of the most serious and continuing threats to aquatic biodiversity in streams and rivers. It is commonly found that both seasonal and subdaily flow patterns are changed below hydropeaking dams due to the multifunctions of the reservoirs such as flood control and water supply. The aim of this paper is to comprehensively quantify the degree of ecologically relevant hydrologic alteration downstream of Gangkouwan Reservoir, focusing particularly on the subdaily flow variation induced by hydropeaking operations. The method is based on the assessment framework set by the indicators of hydrologic alteration (IHA). Hydrologic parameters in groups 1–3 of the IHA were adopted to assess the degree of hydrologic alteration at daily to monthly time scales and based on a daily mean flow data series for both the pre- and post-impact periods. A set of biologically relevant hydrologic parameters based on post-dam hourly flow data, including magnitude and duration of baseflow, magnitude and duration of peaking flows, frequency of peaking flows and ramping rates, were proposed to depict subdaily flow pulses and water condition changes induced by hydropeaking. The results show that Gangkouwan Reservoir made the intra-annual distribution of runoff more consistent, reduced annual peak discharges, and changed the timing of the yearly maximum. The monthly distribution of these hydrologic parameters at the hourly temporal scale provides detailed information on ecologically significant features of the subdaily flow variation induced by hydropeaking operations. This paper provides useful guidance for further investigations on responses of river biota to hydropeaking and provides guidance for suggesting mitigation measures against hydropeaking effects.