Background: Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. Methods: The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. Results: Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. Conclusions: This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.