In light of an increasing trend in fish biology towards using static respirometry techniques without the inclusion of a mixing mechanism and without accurately accounting for the influence of microbial (background) respiration, this paper quantifies the effect of these approaches on the oxygen consumption rates (ṀO2 ) measured from juvenile barramundi Lates calcarifer (mean ± s.e. mass = 20·31 ± 0·81 g) and adult spiny chromis damselfish Acanthochromis polyacanthus (22·03 ± 2·53 g). Background respiration changed consistently and in a sigmoidal manner over time in the treatment with a mixing device (inline recirculation pump), whereas attempts to measure background respiration in the non-mixed treatment yielded highly variable estimates of ṀO2 that were probably artefacts due to the lack of water movement over the oxygen sensor during measurement periods. This had clear consequences when accounting for background respiration in the calculations of fish ṀO2 . Exclusion of a mixing device caused a significantly lower estimate of ṀO2 in both species and reduced the capacity to detect differences between individuals as well as differences within an individual over time. There was evidence to suggest that the magnitude of these effects was dependent on the spontaneous activity levels of the fish, as the difference between mixed and non-mixed treatments was more pronounced for L. calcarifer (sedentary) than for A. polyacanthus (more spontaneously active). It is clear that respirometry set-ups for sedentary species must contain a mixing device to prevent oxygen stratification inside the respirometer. While more active species may provide a higher level of water mixing during respirometry measurements and theoretically reduce the need for a mixing device, the level of mixing cannot be quantified and may change with diurnal cycles in activity. To ensure consistency across studies without relying on fish activity levels, and to enable accurate assessments of background respiration, it is recommended that all respirometry systems should include an appropriate mixing device.