The body temperature of ectotherms can profoundly influence their physiological and ecological performance: but little is known about how the time of testing affects their performance. Although conducting experiments on nocturnal organisms during daylight hours is convenient to the researcher and provides logistic advantages, how this practice affects the performance of species is not well known. Time of day may be particularly relevant for studies on nocturnal reptiles to ensure that the findings are ecologically relevant. Herein, I investigated whether temperature and time of testing affected the locomotor performance of the nocturnal velvet geckos, Oedura lesueurii. I measured locomotor performance of adult females at four different temperatures (20°, 25°, 30° and 35°C) during the day and at night. The sprint speed of geckos was affected by the temperature and time of testing (i.e. night). Females ran fastest at 30°C, and ran faster at the time of day when they are usually active (i.e. night) at all four test temperatures. Geckos made more stops and took longer to complete the racetrack during the diurnal trials than during the nocturnal trials. Circadian rhythms affect performance and the animals are likely to be under strong selection to perform best at the time of day when they are mostly active. My research shows that both temperature and time of the day when animals can influence the locomotor performance of ectotherms. These significant aspects of the experiment design have hitherto been ignored in previous performance studies. Indeed, the organisms’ circadian rhythm and body temperature during experiments on performance should be considered when modelling the effects of increases in temperature on reptile physiology, behaviour, and performance.