In freshwater systems, dissolved oxygen (DO) saturation frequently fluctuates, falling at night and rising during the day in response to respiration and photosynthesis, respectively, of aquatic biota. Low DO (hypoxia) is a common cause of fish kills in freshwater systems around the world. Laboratory studies on responses of fish to fluctuating DO are currently limited, and require techniques that produce a realistic cycle of DO depletion and replacement. Artificial DO-depletion mechanisms frequently used for hypoxia studies may underestimate the field effects of hypoxia on fish because of the lack of the naturally occurring synergistic effect of lower pH, and seldom allow fish to employ behavioural adaptations to hypoxia, such as aquatic surface respiration. We demonstrate proof-of-principle for an alternative method of creating fluctuating hypoxia in an experimental environment, using the natural rhythms of photosynthesis and respiration of aquatic plants to create realistic conditions. A range of volumes of aquatic macrophytes were used alone and in combination with fish to lower DO saturation in sealed freshwater aquaria, and achieved DO saturations as low as 1.3%. This cost-effective method can be deployed over long periods with minimal effort in comparison to traditional methods of DO reduction.