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 °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 ~ 1 µV. 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. Electromagnetic fields (EMF) are generally believed to have no relevant non-thermal effects on cells, tissues, and living organisms 1,2. Only EMF with an excessive strength of > 1.000 kV/m exhibits non-thermal membrane effects such as electroporation 3 or bactericidal microwave exposure 4. Recently, non-thermal effects have been clinically exploited with the tumor-treating field method 5,6 , which applies an EMF at radio frequencies (RF) of 100-300 kHz with a moderate strength of 100-150 V/m. The scientific community considers the risk of such moderate-strength RF-EMF to be negligible, at least with respect to potential hazards caused by powerlines or mobile phones 7-11. Nevertheless, some unresolved observations remain 12,13 , and further research is still recommended 14. However, further specific investigations are only rarely performed because the mechanisms of non-thermal effects remain unknown 15. Oncologists have applied RF technology with similar EMF levels of 200 V/m to cancer treatment using either capacitive (8-30 MHz) or radiative (70-120 MHz) techniques 16. The temperature increase is regarded as the major working mechanism 17. Preclinical data for water bath hyperthermia (WB-HT) have indicated that a temperature of greater than 42 °C is required 18. However, this is rarely achieved in clinical practice, and a temperature achieved in 90% of the target (T 90) of only 39.5-40.5 °C has been correlated with effectiveness 19. Meanwhile, a series of positive randomized trials has shown that RF hyperthermia (RF-HT) improves the effectiveness of radiotherapy or radio-chemotherapy for cervical cancer 20-22. In contrast, extreme whole-body hyperthermia, as the clinical counterpart of WB-HT, using temperatures of ≥ 42 °C has proved to be less effective leading to disappointing results in cancer therapy 23. This indicates that non-thermal effects of RF-EMF do exist. Preclinical studies with animal tumors and cell suspensions 24-26...