BACKGROUND: Long-lasting insecticidal nets (LLINs) are the most sustainable and effective malaria control tool currently available. Global targets are for 80% of the population living in malaria endemic areas to have access to (own) and use a LLIN. However, current access to LLINs in endemic areas is 56% due to system inefficiencies and budget limitations. Thus, cost-effective approaches to maximize access to effective LLINs in endemic areas are required. This study evaluated whether LLINs that had been stored for 5 years under manufacturer's recommended conditions may be optimally effective against Anopheles mosquitoes, to inform malaria control programmes and governments on the periods over which LLINs may be stored between distributions, in an effort to maximize use of available LLINs. METHODS: Standard World Health Organization (WHO) bioassays (cone and tunnel test) were used to evaluate the bio-efficacy and wash resistance of Olyset® and DawaPlus® 2.0 (rebranded Tsara® Soft) LLINs after 5 years of storage at 25 °C to 33.4 °C and 40% to 100% relative humidity. In addition, a small scale Ifakara Ambient Chamber test (I-ACT) was conducted to compare the bio-efficacy of one long stored LLINs to one new LLIN of the same brand, washed or unwashed. LLINs were evaluated using laboratory reared fully susceptible Anopheles gambiae sensu stricto (s.s.) (Ifakara strain) and pyrethroid resistant Anopheles arabiensis (Kingani strain). RESULTS: After 5 years of storage, both unwashed and washed, Olyset® and DawaPlus® 2.0 (Tsara® Soft) LLINs passed WHO bio-efficacy criteria on knockdown (KD60) ≥ 95%, 24-h mortality ≥ 80% and ≥ 90% blood-feeding inhibition in WHO assays against susceptible An. gambiae s.s. DawaPlus® 2.0 LLINs also passed combined WHO bioassay criteria against resistant An. arabiensis. Confirmatory I-ACT tests using whole nets demonstrated that long-stored LLINs showed higher efficacy than new LLINs on both feeding inhibition and mortality endpoints against resistant strains. CONCLUSIONS: Even after long-term storage of around 5 years, both Olyset® and DawaPlus® 2.0 LLINs remain efficacious against susceptible Anopheles mosquitoes at optimal storage range of 25 °C to 33.4 °C for temperature and 40% to 100% relative humidity measured by standard WHO methods. DawaPlus® 2.0 (Tsara® Soft) remained efficacious against resistant strain.