This study aims to investigate the frequency-dependent effects of near-infrared (NIR) transcranial photobiomodulation (tPBM) on human cortical excitability and fine motor performance. A cohort of 20 healthy adult volunteers (aged 18-35 years) will participate in a randomized, double-blind, sham-controlled, single-center study utilizing a within-subject, five-arm crossover design. Each participant will undergo five distinct experimental conditions separated by a strict washout period: active Continuous Wave (CW) tPBM, active pulsed tPBM at three neural oscillation frequencies (10 Hz, 40 Hz, and 100 Hz), and an inactive Sham comparator. All interventions will be delivered using a clinical-grade near-infrared device.

To isolate the specific effects of pulse frequency from overall energy delivery, the total energy dose (cumulative Joules) and total exposure time will be held perfectly constant across all active arms. The pulsed conditions will keeps the average irradiance and cumulative fluence identical to the continuous wave mode. Cortical excitability will be assessed via single- and paired-pulse Transcranial Magnetic Stimulation (TMS) protocols over the primary motor cortex (M1). Fine motor performance will be quantified using a smartphone-based Finger Tapping Test (FTT). Safety and systemic parameters will be tracked using the Systematic Assessment for Treatment Emergent Events - Systematic Inquiry (SAFTEE-SI) and vital signs. The central hypothesis is that distinct modulation frequencies will differentially influence corticospinal excitability and motor execution, independent of the total energy delivered.

Transcranial photobiomodulation (tPBM) has emerged as a promising non-invasive tool to modulate neural activity through the absorption of near-infrared (NIR) photons by mitochondrial cytochrome c oxidase (CCO). However, whether the biological effects on the human cortex are driven solely by cumulative energy delivery or are significantly modulated by pulse frequency remains a critical question in neurophysiology. This study implements a rigorous within-subject crossover design to systematically isolate the effects of pulse frequency from total dosimetric parameters. Participants and Screening: A sample of 20 healthy volunteers (aged 18-35) will be recruited. Potential candidates will undergo a strict screening protocol to ensure safety and baseline homogeneity. Exclusion criteria include any contraindications to magnetic fields assessed by the Transcranial Magnetic Stimulation Adult Safety Screen (TASS; Rossi et al., 2021), psychiatric conditions according to DSM-5, a history of neurological disorders, the use of psychotropic medications within the last 12 months, or prior participation in an interventional neuromodulation study within the preceding 6 months. Experimental Design & Intervention: Enrolled participants will complete five experimental sessions in a randomized, counterbalanced order to eliminate carryover or sequence effects, separated by a mandatory washout period to ensure the return of cortical excitability to baseline. In each session, a clinical-grade near-infrared system will be applied over the primary motor cortex (M1) hot spot. The five experimental arms consist of:

Continuous Wave (CW) active tPBM at baseline irradiance. 10 Hz near-infrared active tPBM. 40 Hz near-infrared active tPBM. 100 Hz near-infrared active tPBM. Sham Comparator (inactive control). Physical Calibration and Blinding: To ensure flawless dosimetric matching, total energy dose (Joules) and application duration will be kept constant across all active arms. The active pulsed interventions (10 Hz, 40 Hz, and 100 Hz) will utilize a 50% duty cycle with a peak irradiance exactly twice that of the Continuous Wave (CW) mode, thereby ensuring that the average irradiance (mW/cm2) and cumulative fluence (J/cm2) remain strictly identical to the CW. For the Sham condition, the device will emit a minimal, sub-therapeutic visual output of visible red light. This serves as a visual guide mimic to maintain complete participant blinding without transferring active near-infrared photons to the cortex (0 J active NIR dose). Double-blinding will be enforced for the participant and investigator. Alphanumeric codes will mask the active protocols on the user interface, and participants will wear opaque safety goggles. Outcome Measures: Multiple neurophysiological, behavioral, and safety endpoints will be collected immediately pre-intervention (baseline) and post-intervention. Neurophysiology (TMS): Motor Evoked Potential (MEP) amplitude (primary excitability index); Intracortical Facilitation (ICF); Short-Interval Intracortical Inhibition (SICI); and Cortical Silent Period (CSP). Behavioral Performance (FTT): Evaluated via a dedicated Android application tracking total number of taps, variability of the inter-tap interval (vITI), spatial resultant sum (Σ||Δr||), and the 95% confidence ellipse area (X,Y).

Safety and Hemodynamics: Systemic tolerability will be recorded via the Systematic Assessment for Treatment Emergent Events - Systematic Inquiry (SAFTEE-SI). Hemodynamic variations will be closely monitored through independent analyses of Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), and Heart Rate (HR). Data will be processed using Linear Mixed-Effects Models to account for the repeated-measures structure of the crossover design.

Inclusion Criteria:

• Voluntary Participation: Participants who are clear of their cognitive faculties, capable of understanding all experimental procedures, and who provide written informed consent prior to enrollment.
• Neurologically and Psychiatrically Healthy Status: Individuals with no current or prior history of neurological, neurodevelopmental, or psychiatric conditions;
• Age Range: Young adult volunteers aged between 18 and 35 years.

Exclusion Criteria:

• Use of any continuous psychotropic medication within the past 12 months.
• Any current psychiatric diagnosis based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5).
• Diagnosis of any neurological disorder capable of altering cortical excitability (e.g., seizures/epilepsy, cerebrovascular accidents/stroke, brain tumors).
• Any clinical, psychological, or social condition that, in the investigator's opinion, places the participant at an increased risk, compromises participant safety, or precludes full compliance and successful completion of the study protocol.
• Presence of any intracranial devices or implants, including cochlear implants and aneurysm clips.
• Severe or uncompensated systemic medical illness that could interfere with study participation or confound physiological outcomes.
• Participation in any other interventional neuromodulation study within the preceding 6 months.

To evaluate the integrity of the blinding procedure, a Blinding Index assessment will be conducted at the end of each participant's final experimental session. Participants and the primary investigator will complete a forced-choice questionnaire to guess which intervention (Active or Sham) was administered in each of the five sessions. The success of the blinding protocol will be statistically confirmed if the distribution of correct guesses does not significantly deviate from random chance (p > 0.05 via chi-square analysis).