Objective: This study aimed to estimate the influence of the duration of mobile phone use on heart rate variability (HRV) in healthy individuals. Methods: One hundred forty-eight individuals without any established systemic disease and who had undergone 24-h ambulatory ECG monitoring were included in the case-control study. All the individuals had been using mobile phones for more than 10 years. Three-channel 24-h Holter monitor- ing was performed to derive the mean heart rate, standard deviation of normal NN intervals (SDNN), standard deviation of 5-min (m) mean NN intervals (SDANN), the proportion of NN50 divided by the total number of NNs (pNN50), the root mean square differences of successive NN intervals (RMSSD), high (HF)-, low (LF)-, very low (VLF)-frequency power, total power components, and the LF/HF ratio. Individuals were divided into four groups according to their duration of mobile phone use [no mobile phone use (Control group), <30 min/day (Group 1), 30–60 min/day (Group 2), and >60 min/day (Group 3)]. Results: All the groups had similar features with regard to demographic and clinical characteristics. No significant arrhythmias were observed in any of the groups. The LF/HF ratio was higher, whereas the SDNN, SDANN, RMSSD, and pNN50 values were lower in the study groups than in the control group (p<0.05). No significant differences were identified among groups with respect to heart rate, VLF, and total power values (p>0.05). Conclusion: In this study, it was shown that the duration of mobile phone use may affect the autonomic balance in healthy subjects. The elec- tromagnetic field created by mobile phone use may induce HRV changes in the long term. (Anatol J Cardiol 2016; 16: 833-8)