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New classes of long-lasting insecticidal nets (LLINs), have been recommended by the World Health Organization (WHO) to control malaria vectors resistant to pyrethroid insecticides. This study was nested in a large-scale cluster-randomized controlled trial conducted (cRCT) in Tanzania. A series of experimental hut trials (EHTs) aimed to evaluate the bio-efficacy of trial LLINs on mosquito indicators most pertinent to malaria transmission over 3 years of use in the community in order to better understand the outcomes of the cRCT. The following field-collected LLINs were assessed: 1) Olyset Plus (combining piperonyl butoxide synergist and permethrin), 2) Interceptor G2 (chlorfenapyr and alpha-cypermethrin), 3) Royal Guard (pyriproxyfen and alpha-cypermethrin), 4) Interceptor (alpha-cypermethrin only) conducted in parallel with 5) a new Interceptor, and 6) an untreated net. Thirty nets of each type were withdrawn from the community at 12, 24, and 36 months after distribution and used for the EHTs. Pre-specified outcomes were 72-hour mortality for Interceptor G2, 24-hour mortality for Olyset Plus, and fertility based on egg development stage for Royal Guard. Overall, Interceptor G2 LLINs induced higher 72-hour mortality compared to standard LLINs of the same age up to12 months (44% vs 21%, OR: 3.5, 95% CI: 1.9-6.6, p-value < 0.001), and 24-hour mortality was only significantly higher in Olyset Plus when new (OR: 13.6, 95%CI: 4.4-41.3, p-value < 0.001) compared to standard LLINs but not at 12 months (17% vs 13%; OR: 2.1, 95% CI: 1.0-4.3; p-value = 0.112). A small, non-significant effect of pyriproxyfen on Anopheles fertility was observed for Royal Guard up to 12 months (75% vs 98%, OR: 1.1, 95% CI: 0.0-24.9, p-value = 0.951). There was no evidence of a difference in the main outcomes for any of the new classes of LLINs at 24 and 36 months compared to standard LLINs. Interceptor G2 LLINs showed superior bio-efficacy compared to standard LLINs only up to 12 months, and the effect of Olyset Plus was observed when new for all species and 12 months for An. gambiae s.l. only. The pyriproxyfen component of Royal Guard had a short and limited effect on fertility. The decrease in effectiveness of Olyset Plus and Royal Guard LLINs in the EHTs aligns with findings from the cRCT, whereas efficacy of Interceptor G2 lasted for a longer period in the cRCT compared to the EHT. Further investigations are needed to understand the complete scope of chlorfenapyr mode of action. Additional EHT in various contexts will help confirm the residual efficacy of the dual active ingredient LLINs and support the development of longer-lasting nets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)