Frequency-Dependent Effects of Transcranial Photobiomodulation With Same Peak Irradiance on Cortical Excitability and Fine Motor Performance

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) tPBM - NIR light delivered continuously at baseline peak irradiance, 1x total cumulative energy dose; 10 Hz Pulsed tPBM - NIR light pulsed at 10 Hz with identical peak irradiance as CW and a 50% duty cycle, 0.5x total dose relative to CW; 40 Hz Pulsed tPBM - NIR light pulsed at 40 Hz with identical peak irradiance as CW and a 50% duty cycle, 0.5x total dose relative to CW; 100 Hz Pulsed tPBM - NIR light pulsed at 100 Hz with identical peak irradiance as CW and a 50% duty cycle, 0.5x total dose relative to CW; sham Control - a dedicated low-intensity red light photobiomodulation device emitting visible red light at sub-therapeutic levels to maintain participant blinding. 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.

• Transcranial Photobiomodulation (Combination product), also known as Low Level Laser Therapy: Transcranial photobiomodulation (tPBM) is a non-invasive, non-thermal neuromodulatory modality that utilizes low-power coherent (laser) or non-coherent (light-emitting diodes, LEDs) light sources within the red (lambda = 600-700 nm) and near-infrared (NIR; lambda = 700-1100 nm) spectral windows to modulate cortical function. Structurally tailored to penetrate superficial anatomical barriers-including the scalp, skull, and meninges-tPBM delivers photons directly to the cerebral cortex.

Primary outcomes

• Motor Evoked Potential (MEP) Amplitude - TMS [Immediately before the intervention (pre-intervention) and 20 minutes after the intervention (post-intervention)]: Single-pulse TMS will be applied over the primary motor cortex (M1) hotspot to elicit Motor Evoked Potentials (MEPs) recorded via electromyography (EMG) from the target muscle (e.g., first dorsal interosseous, FDI). Peak-to-peak MEP amplitude (mV) will be measured at a stimulation intensity adjusted to evoke a baseline response of approximately 1 mV. This outcome reflects overall baseline corticospinal excitability and its modulation following the tPBM protocol.
• Intracortical Facilitation (ICF) - TMS [Immediately before the intervention (pre-intervention) and 20 minutes after the intervention (post-intervention)]: Evaluated using a paired-pulse TMS protocol consisting of a subthreshold conditioning stimulus (80% of resting motor threshold, RMT) followed by a suprathreshold test stimulus (120% RMT) at a long interstimulus interval (ISI) of 10 ms. The outcome is expressed as the ratio of the conditioned MEP amplitude to the unconditioned test MEP amplitude. ICF is primarily mediated by cortical glutamatergic circuits and NMDA receptor activity.
• Short-Interval Intracortical Inhibition (SICI) - TMS [Immediately before the intervention (pre-intervention) and 20 minutes after the intervention (post-intervention)]: Assessed via a paired-pulse TMS paradigm using a subthreshold conditioning stimulus (80% RMT) followed by a suprathreshold test stimulus (120% RMT) at a short interstimulus interval (ISI) of 3 ms. The resulting SICI value is quantified as the percentage of inhibition of the conditioned MEP relative to the unconditioned test MEP. This parameter indexes local intracortical inhibitory interneuron activity mediated by GABA_A receptors.
• Cortical Silent Period (CSP) - TMS [Immediately before the intervention (pre-intervention) and 20 minutes after the intervention (post-intervention)]: Induced by applying a single suprathreshold TMS pulse (120% RMT) over the M1 hotspot while the participant maintains a stable, isometric voluntary contraction of the target muscle (e.g., 20% of maximum voluntary contraction). The CSP duration (ms) is measured from the onset of the MEP to the return of rectified background EMG activity. CSP duration provides a precise marker of interhemispheric and intracortical inhibition mediated by GABA_B receptors.

Secondary outcomes

• Total Number of Taps - FTT [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: The cumulative count of valid screen contacts executed by the participant's index finger within a fixed, standardized testing interval (e.g., 30 seconds). This metric serves as a behavioral index of maximal motor execution speed and tapping frequency.
• Variability of the Inter-Tap Interval (vITI) - FTT [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: Calculated as the standard deviation (or coefficient of variation) of the temporal intervals between consecutive screen contacts (in milliseconds). This outcome quantifies the temporal rhythmic precision and stability of the central motor program.
• Spatial Resultant Sum (Σ||Δr||) - FTT [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: The cumulative Euclidean distance calculated across all sequential tap coordinates on the 2D Android screen interface. This parameter reflects spatial dispersion and motor drift, tracking the continuous precision of the targeted finger-pointing trajectory.
• 95% Confidence Ellipse Area (X,Y) - FTT [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: Computed as the total geometric surface area (in squared millimeters, mm2) of the bivariate error ellipse that encompasses 95% of the coordinates of all performed taps on the horizontal (X) and vertical (Y) axes. This spatial metric quantifies overall motor accuracy and targeting consistency.
• Adverse Events and Tolerability (SAFTEE-SI) [Baseline, before all interventions and one week after the last intervention]: The safety and tolerability profile of the combined neuromodulation protocol will be systematically evaluated using the Systematic Assessment for Treatment Emergent Events - Systematic Inquiry (SAFTEE-SI). This structured instrument will track the incidence, severity, and potential causal relationship of any somatic, neurological, or behavioral symptoms (e.g., headache, scalp discomfort, fatigue, dizziness, or localized thermal sensations) emerging during or after stimulation.

Other outcomes

• Systolic Blood Pressure (SBP) [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: Systemic systolic blood pressure will be quantified in millimeters of mercury (mmHg) using a calibrated, digital oscillometric upper-arm sphygmomanometer. Measurements will be recorded under standardized resting conditions at pre-intervention (baseline) and immediately post-intervention across all five crossover arms to monitor potential acute alterations in peak arterial pressure induced by transcranial photobiomodulation.
• Diastolic Blood Pressure (DBP) [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: Systemic diastolic blood pressure will be quantified in millimeters of mercury (mmHg) using the same automated oscillometric system. Measurements will be recorded concurrently with the systolic parameters at baseline and immediately post-intervention across all five crossover sessions to evaluate potential shifts in minimum arterial pressure during cardiac relaxation.
• Heart Rate (HR) [Immediately before the intervention (pre-intervention) and immediately after the intervention (post-intervention)]: Heart rate will be quantified in beats per minute (bpm) concurrently with the blood pressure evaluation using the same automated digital system. This parameter serves as an acute physiological indicator of autonomic nervous system balance (sympathovagal tone) before and after the application of continuous or sinusoidal pulsed photobiomodulation protocols.

Sex: All, ages: 18 Years to 35 Years, accepts healthy volunteers

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.

• Marco A Caldieraro, MD PhD, Principal investigator, Federal University of Health Science of Porto Alegre