Following the recent deployment of fifth-generation (5G) radio frequencies, several questions about their health impacts have been raised. Due to the lack of experimental research on this subject, the current study aimed to investigate the bio-physiological effects of a generated 3.5 GHz frequency. For this purpose, the wake electroencephalograms (EEG) of 34 healthy volunteers were explored during two “real” and “sham” exposure sessions. The electromagnetic fields were antenna-emitted in an electrically shielded room and had an electrical field root-mean-square intensity of 2 V/m, corresponding to the current outdoor exposure levels. The sessions were a maximum of one week apart, and both contained an exposure period of approximately 26 min and were followed by a post-exposure period of 17 min. The power spectral densities (PSDs) of the beta, alpha, theta, and delta bands were then computed and corrected based on an EEG baseline period. This was acquired for 17 min before the subsequent phases were recorded under two separate conditions: eyes open (EO) and eyes closed (EC). A statistical analysis showed an overall non-significant change in the studied brain waves, except for a few electrodes in the alpha, theta, and delta spectra. This change was translated into an increase or decrease in the PSDs, in response to the EO and EC conditions. In conclusion, this studhy showed that 3.5 GHz exposure, within the regulatory levels and exposure parameters used in this protocol, did not affect brain activity in healthy young adults. Moreover, to our knowledge, this was the first laboratory-controlled human EEG study on 5G effects. It attempted to address society’s current concern about the impact of 5G exposure on human health at environmental levels.