Despite effects of dams and water extraction on river hydrology and consequences of aquatic ecosystems being broadly appreciated, empirical evidence is lacking for many regions (e.g. subtropics). Evidence is necessary to determine (i) the circumstances where environmental flows are necessary to protect or improve ecological processes and (ii) what hydrological events are required to achieve those ecological characteristics. Here, temporal variation in the size structure of two small-bodied fish species with contrasting ecological characteristics (Australian smelt, Cox's gudgeon) was compared between two pairs of unregulated and regulated rivers in subtropical Australia. Frequency of in-channel flow pulses in each regulated river was lower compared to paired unregulated rivers. An experimental flow pulse was delivered to one regulated river to assess the ecological outcomes of in-channel flow pulses and inform future decisions about the use of environmental water allocations. Temporal changes in the population size structure of both species were similar between unregulated and regulated rivers and showed no response to the experimental pulse. While the experimental flow briefly suppressed in-stream temperature, changes in temperature were not beyond the thresholds at which spawning occurs or the thermal tolerances for either species. Similar population structure between unregulated and regulated rivers can be attributed to the magnitude of flow regulation being insufficient to alter the physico-chemical conditions, habitat and trophic mechanisms supporting population dynamics during the study period. This suggests current regulated hydrology is protective of local populations of Australian smelt and Cox's gudgeon in these study rivers.