Moisture in asphalt concrete (AC) pavements does not only cause distresses like potholes, ravelling, etc. but also exacerbates distresses like fatigue and rutting. Moisture susceptibility of AC mixtures is usually characterized using the Modified Lottman Test (AASHTO T283) adopted in Superpave mix design. This test has been observed to provide a good prediction of moisture susceptibility of AC mixtures. However, it does not provide insight on how moisture affects the fatigue characteristics of AC mixtures. Therefore, there is a need for a test procedure that can characterize the effect of moisture on the fatigue behaviour of AC mixtures. In this study, changes in fatigue characteristics after moisture conditioning were evaluating by conducting four-point beam (4 PB) fatigue tests on dry and moisture conditioned beam specimens. For this purpose, specimens of AC mixed with two different types of binders with target air voids of 4 ± 0.5% were prepared. These specimens were subjected to partial vacuum saturation by submerging completely in water and applying vacuum pressure to the system. The fatigue tests were conducted at four different strain amplitudes of 200, 400, 600 and 800 micro-strains. The results were then compared with the fatigue tests results obtained with dry beam specimens. Test results show that the conditioning reduces both the fatigue life and the initial flexural stiffness of the beam specimens. Moreover, it was also observed that the binder type plays a significant role in the degree of saturation. © 2020, Springer Nature Switzerland AG.