1. Although laboratory studies of the behaviour of aquatic macroinvertebrates are common, there has been little critical evaluation of the importance of test conditions to them. We used a common Australian leptophlebiid mayfly, Nousia sp., to investigate responses to light, wavelength of light, presence or absence of cover and still or flowing water.2. Nousia sp. showed substantial qualitative differences in behaviour, as measured by movement, when there was no refuge (in the form of a crevice beneath a tile) present in the experimental arena.3. We found no evidence of diel periodicity in activity in Nousia sp.4. Nousia sp. did not respond to infra‐red, red or green light at a flux density of 18–19 μmol m–2 s–1.5. Nymphs were three times more likely to remain stationary in flowing water (mean velocity 0.10 m s–1) than in still water.6. We conclude that generalized assumptions about test conditions for experiments designed to quantify laboratory behaviour in benthic macroinvertebrates are unjustified and that evaluation of the individual requirements of test species should be conducted routinely.