Background/Question/Methods The influence of climate on the intrinsic probability of malaria transmission is usually explored using estimates of mean monthly (sometimes yearly) temperature. However, temperatures fluctuate throughout the diurnal cycle and hence, small-bodied insects, such as mosquitoes, rarely experience ‘mean' conditions. Here we combine novel empirical data and a thermodynamic malaria development model to demonstrate that temperature fluctuation can alter the incubation period of the parasite within the mosquito considerably. This incubation period is the most influential parameter determining the intensity of malaria transmission. Results/Conclusions We find that, in general, temperature fluctuation reduces the impact of increasing mean temperatures. Specifically, we show that diurnal temperature fluctuation around means >21oC increases the incubation period, whereas fluctuation around means of <21oC, reduces the incubation period, compared with constant temperatures. These effects suggest that by ignoring fluctuation, we may currently be overestimating malaria risk in warmer environments, and underestimating risk in cooler environments. We further show that temperature fluctuation has equivalent effects on the rate of larval development and also impacts the probability of mosquitoes becoming infected with malaria following a blood meal from an infected host. Such effects of short-term temperature fluctuations have not previously been considered but appear central to understanding current malaria transmission and for evaluating consequences of future climate change.