Given the large number of mobile phone users and the increasing exposure to radiofrequency electromagnetic field (RF-EMF) worldwide, we aimed to study the effect of RF-EMF related to mobile phones on heart rate variability (HRV). Twenty-six healthy young adults participated in two experimental sessions with a double-blind, randomized and counter-balanced crossover design. During each session, participants were exposed for 26 min to a sham or real 900 MHz RF-EMF, generated by a commercial dual-band Global System for Mobile technology (GSM) mobile phone. We recorded an electrocardiogram at rest during the exposure. We evaluated HRV by time- and frequency-domain analysis. Evaluation of time-domain HRV parameters revealed a statistically significant increase of the standard deviation of interbeat intervals (SDNN) during the real exposure. Other time-domain parameters were not affected. Analysis in the frequency-domain demonstrated that total spectral power and low-frequency band (LF) absolute power were significantly increased during exposure (p = .046 and p = .043, respectively). However, other parameters were not affected. In conclusion, it seems that most HRV parameters were not affected by GSM signal exposure in our study. The weak effect observed on HRV frequency-domain is likely to represent a random occurrence rather than a real effect.