Purpose: To investigate the effects of interfractional variation, such as anatomical changes and setup errors, on dose delivery during treatment for prostate cancer (PC) and head and neck cancer (HNC) by courses of volumetric modulated arc therapy (VMAT) aided by on-treatment electronic portal imaging device (EPID) images.Methods: Seven patients with PC and 20 patients with HNC who had received VMAT participated in this study. After obtaining photon fluence at the position of the EPID for each treatment arc from on-treatment integrated EPID images, we calculated the differences between the fluence for the first fraction and each subsequent fraction for each arc. The passing rates were investigated based on a tolerance level of 3% of the maximum fluence during the treatment courses and the correlations between the passing rates and anatomical changes.Results: In PC, the median and lowest passing rates were 99.8% and 95.2%, respectively. No correlations between passing rates and interfractional variation were found. In HNC, the median passing rate of all fractions was 93.0%, and the lowest passing rate was 79.6% during the 35th fraction. Spearman's correlation coefficients between the passing rates and changes in weight or neck volume were − 0.77 and − 0.74, respectively.Conclusions: Analyses of the on-treatment EPID images facilitates estimates of the interfractional anatomical variation in HNC patients during VMAT and thus improves assessments of the need for re-planning or adaptive strategies and the timing thereof.