Feasibility study of 40 Hz trans-eyelid visual stimulation combined with SSVEP technology in healthy adults for neuromodulation

Abstract

Forty-hertz visual stimulation, within the gamma frequency band, has been shown to entrain cortical oscillations and influence neurobiological processes linked to Alzheimer's disease (AD) pathology, including enhanced synaptic plasticity, activation of microglia-mediated clearance, reduction of amyloid-(3 and tau burden, and restoration of network synchrony. While these mechanisms highlight therapeutic potential, most human studies lack online electrophysiological confirmation of entrainment and rely on eyes-open protocols that may cause glare, fatigue, and limit use in sensitive populations. We developed and evaluated a closed-eye steady-state visual evoked potential (SSVEP) protocol that leverages partial visible-light transmission through the eyelids to stimulate the visual cortex without direct gaze. Healthy participants received flicker stimulation at four frequencies (4, 17, 25, 40 Hz) and five colors (white, red, green, blue, yellow), as well as SSVEP under four different distances (0, 10, 30, 60 cm), under both eyes-open and eyes-closed conditions. EEG recordings were analyzed for signal-tonoise ratio (SNR) and phase locking value (PLV) to quantify the strength of cortical entrainment. Closed-eye 40 Hz stimulation consistently evoked frequency-locked SSVEPs with SNR comparable to eyes-open trials. White and green light elicited the strongest responses overall. While exhibiting lower amplitudes compared to lower frequencies, 40 Hz stimulation produced reliable phase-locked entrainment (as indicated by PLV) consistent with gamma-frequency mechanisms that support synaptic and network-level neuromodulation. This study provides a systematic validation of closed-eye 40 Hz SSVEP stimulation as a safe, comfortable, and effective method for verifying online entrainment. Combining physiological relevance with practical delivery offers a quantitative strategy for developing neuromodulation protocols.