A fasting period prior to non-circulative virus acquisition has been shown to increase the rate of transmission by aphids. However, this effect has only been studied for a few virus–vector combinations, and there are contradictory results in the literature as to the role of fasting on virus acquisition. We analysed the influence of fasting on the transmission of three non-circulative viruses, Cucumber mosaic virus, Zucchini yellow mosaic virus and Cauliflower mosaic virus, by two aphid vector species: Myzus persicae Sulzer (Hemiptera: Aphididae) and Aphis gossypii Glover (Hemiptera: Aphididae). All variables tested, including the virus species and isolate, and the species of aphid, influenced the effect of a fasting period on virus transmission efficiency. Furthermore, when aphids were subjected to an overnight feeding period on a sucrose solution, the fasting effect disappeared and the probing behaviour of these aphids was markedly different to plant-reared aphids. The electrical penetration graph (EPG) technique revealed that fasting altered the probing behaviour of M. persicae and A. gossypii, with fasted aphids beginning to feed sooner and having a significantly longer first intracellular puncture, measured as a potential drop. Significantly longer sub-phase II-3 of the potential drop and more archlets during this sub-phase were also observed for fasted aphids of both species. However, these behavioural changes were not predictive of increasing virus transmission following a fasting period. The impacts of pre-acquisition fasting on aphid probing behaviour and on the mechanisms of non-circulative virus transmission are discussed.