Introduction: Acute smartphone use can decrease cognitive and physical performance, likely due to the induction of mental fatigue. To date, no study has used electrophysiological markers to support this hypothesis. Mental fatigue is known to induce changes in brain activity (e.g., increase in theta and alpha power), cardiac activity (e.g., increase in heart rate variability - HRV) and ocular activity (e.g., increase in pupil diameter), as well as increasing feelings of tiredness and lack of energy. The aim of this study was to investigate the effects of acute smartphone use on electroencephalography, pupillometry, and HRV responses to objectively identify the presence of mental fatigue. Methods: Eighteen participants performed a psychomotor vigilance task (PVT) before and after 45-min of smartphone use. Feelings of fatigue, and sleepiness, were assessed with visual analog scales before and after smartphone use. Brain oscillations, HRV, and pupil diameter were recorded 2-min at rest before and after, as well as during smartphone use. Evoked-related potentials (N200 and P300) were investigated during the PVT. Results: Acute smartphone use increased feelings of fatigue (t17 = -4.567, p < .001), and sleepiness (t17 = -3.585, p = .002). During smartphone use, theta power increased on frontal (t17 = -2.470, p = .024) and centromedial (t17 = -2.353, p = .031) regions, without any changes in pupil diameter and HRV. During the rest period after smartphone use, alpha power increased in the central left (t17=-2.227, p=.040), central right (t17 = -2.604, p = .018), and all parietal regions (p < .033|). HRV high frequencies decreased (t17 = 2.818, p = .013), but pupil diameter did not change. Reaction times increased after smartphone use (t17 = -2.335, p = .032), while no significant effects were reported on N200 and P300. Conclusion: Forty-five minutes of smartphone use induced mental fatigue, subjectively evidenced by an increased feeling of fatigue, and objectively by a decreased attentional performance. The increase in theta power during smartphone use and a decrease in HRV high frequencies after smartphone could reflect the presence of mental fatigue. However, due to the concomitant increase in sleepiness, these electrophysiological changes may not be specific to the induction of mental fatigue. Future studies should explore the dissociation between feelings of fatigue and sleepiness to validate these changes as markers of mental fatigue. Further investigations are needed to obtain reliable electrophysiological markers of mental fatigue after acute smartphone use.