Water managers need quantitative information on the effects of hydrologic alteration on aquatic biota to guide ecologically sensitive water management strategies such as water releases from dams. A key gap in the global research literature is determining whether low levels of hydrologic alteration have significant effects on fish populations and assemblage characteristics. This study quantified patterns of fish response to flow regime alteration in a sub-tropical region where many rivers have regulated flow regimes but 57% of ecologically relevant flow metrics have changed by <20%. We tested for flow regulation effects on 17 (univariate and multivariate) response variables representing fish population abundance and assemblage characteristics using a field design based on the environmental flow assessment frame-work known as ELOHA (Ecological Limits of Hydrologic Alteration). Ecological response variables that are readily quantified and sensitive to variation and alteration in flow regimes are critical to the application of environmental flow frameworks such as ELOHA. In this study only three of 17 response variables rep-resenting fish population abundance and assemblage attributes showed significant differences between regulated and unregulated reaches (densities of both Pseudomugil signifier and Melanotaenia duboulayi, and fish assemblage composition). Effects associated with flow regulation were most evident where his-torically intermittent flow regimes have become more perennial as a consequence of managed water releases from dams. Our study provides positive evidence that dams and regulated flow regimes can be managed with sensitivity such that there are few significant changes in populations of most fish species, and little change in fish assemblage characteristics. However, it must be cautioned that the magnitude of flow regime alteration may interact with the duration of exposure (i.e. years to decades) such that other ecological impacts emerge over time as species and assemblages adjust to altered flow regimes.