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Background: The WHO cone bioassay is routinely used to evaluate the bioefficacy of insecticide-treated nets (ITNs) for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. We investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. Methods: Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and piperonyl butoxide (PBO)) ITNs, using laboratory-reared pyrethroid-resistant Anopheles funestus sensu stricto (FUMOZ strain) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO-recommended test parameters, with one variable changed in each bioassay: (i) environmental temperature during exposure: 22–23 °C, 26–27 °C, 29–30 °C and 32–33 °C; (ii) feeding regimen before exposure: sugar starved for 6 h, blood-fed or sugar-fed; and (iii) mosquito density per cone: 5, 10, 15 and 20 mosquitoes. For each test, 15 net samples per treatment arm were tested with four cones per sample (N = 60). Mortality after 24, 48 and 72 h post-exposure to ITNs was recorded. Results: There was a notable influence of temperature, feeding status and mosquito density on An. funestus mortality for both types of ITNs. Mortality at 24 h post-exposure was significantly higher at 32–33 °C than at 26–27 °C for both the MAGNet [19.33% vs 7%; odds ratio (OR): 3.96, 95% confidence interval (CI): 1.99–7.87, P < 0.001] and Veeralin (91% vs 47.33%; OR: 22.20, 95% CI: 11.45–43.05, P < 0.001) ITNs. Mosquito feeding status influenced the observed mortality. Relative to sugar-fed mosquitoes, The MAGNet ITNs induced higher mortality among blood-fed mosquitoes (7% vs 3%; OR: 2.23, 95% CI: 0.94–5.27, P = 0.068) and significantly higher mortality among starved mosquitoes (8% vs 3%, OR: 2.88, 95% CI: 1.25–6.63, P = 0.013); in comparison, the Veeralin ITNs showed significantly lower mortality among blood-fed mosquitoes (43% vs 57%; OR: 0.56, 95% CI: 0.38–0.81, P = 0.002) and no difference for starved mosquitoes (58% vs 57%; OR: 1.05, 95% CI: 0.72–1.51, P = 0.816). Mortality significantly increased with increasing mosquito density for both the MAGNet (e.g. 5 vs 10 mosquitoes: 7% vs 12%; OR: 1.81, 95% CI: 1.03–3.20, P = 0.040) and Veeralin (e.g. 5 vs 10 mosquitoes: 58% vs 71%; OR 2.06, 95% CI: 1.24–3.42, P = 0.005) ITNs. Conclusions: The results of this study highlight that the testing parameters temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities. [ABSTRACT FROM AUTHOR]