We explored the non-thermal effects of radiofrequency (RF) electromagnetic fields and established a theoretical framework to elucidate their electrophysiological mechanisms. In experiments, we used a preclinical treatment device to treat the human colon cancer cell lines HT-29 and SW480 with either water bath heating (WB-HT) or 13.56 MHz RF hyperthermia (RF-HT) at 42 degrees C for 60 min and analyzed the proliferation and clonogenicity. We elaborated an electrical model for cell membranes and ion channels and estimated the resulting ion fluxes. The results showed that, for both cell lines, using RF-HT significantly reduced proliferation and clonogenicity compared to WB-HT. According to our model, the RF electric field component was rectified and smoothed in the direction of the channel, which resulted in a DC voltage of similar to 1 mu V. This may induce ion fluxes that can potentially cause relevant disequilibrium of most ions. Therefore, RF-HT creates additional non-thermal effects in association with significant ion fluxes. Increasing the understanding of these effects can help improve cancer therapy.