Background Long COVID, also known as Post-COVID Condition (PCC) or Post-Acute Sequelae of
COVID-19 (PASC), is characterized by persistent symptoms following SARS-CoV-2 infection
without an alternative explanation. Fatigue and cognitive dysfunction are among the most
common and disabling complaints, with substantial effects on daily functioning, work
participation, and quality of life. Converging evidence from neuroimaging and fluid
biomarkers points to altered cerebral perfusion, disrupted functional connectivity, and
neuroinflammatory processes in at least a subset of people with PASC.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation
technique that can modulate cortical excitability and large-scale networks, with
downstream effects on cerebral blood flow, connectivity and inflammatory signaling.
Small, uncontrolled studies in PASC and related fatigue conditions suggest potential
benefits of rTMS for fatigue and cognition, but placebo-controlled evidence in PASC is
lacking and prior studies have used relatively few sessions. The present trial addresses
this gap by testing a functional magnetic resonance (fMRI)-guided rTMS protocol in a
randomized, double-blind design, while characterizing neurobiological mechanisms of
change.

Objectives The primary objective is to determine whether high-frequency (10 Hz) rTMS
reduces fatigue severity in adults with PASC compared with sham stimulation. Secondary
objectives are to evaluate effects on physical and cognitive functioning,
patient-reported outcome measures (e.g., mood, sleep, and quality of life), to quantify
rTMS-related changes in neuroimaging and blood-based biomarkers reflecting neuronal
integrity, cerebral perfusion, and (neuro)inflammation, and to examine whether these
biomarkers can predict symptom improvement.

Design and procedures This is a single-center, randomized, double-blind, sham-controlled
clinical trial. Sixty-six adults with PASC characterized by severe fatigue and cognitive
complaints will be enrolled through the Post-COVID Network Netherlands. After baseline
assessments, participants are randomized 1:1 to active rTMS or sham rTMS using block
randomization implemented in Castor EDC with allocation concealment; participants and
outcome assessors are blinded.

Treatment is delivered four times per week for six consecutive weeks (24 sessions). A
minimum effective dose of 16 sessions applies when burden needs to be reduced. Outcome
assessments are conducted within two weeks before the treatment at baseline (T0), within
two weeks after the intervention period (T1), and at follow-up three months (T2) and six
months (T3) after treatment to evaluate long-term effects. During the six-week
intervention period a brief subset of patient-reported measures is collected weekly to
monitor symptom trajectories and adverse effects.

Neuroimaging and blood sampling are obtained at T0 and T1. The MRI protocol (3T Siemens
VIDA) includes structural, perfusion (ASL), resting-state and task-based fMRI (Tower of
London), spectroscopy (MRS), and conventional FLAIR/SWI. Blood-derived biomarkers (e.g.,
NfL, GFAP, brain-derived tau, IL-6/IL-1/TNF-α, CCL11, BDNF) are assayed from EDTA plasma
with standardized handling and storage prior to batch analysis. Actigraphy is assessed
over 8 days prior to treatment and 8 days following treatment, and heart-rate variability
is assessed during 4 nights and 4 times directly after waking for 5 minutes 2 weeks
before treatment and directly after treatment.

Interventions Active treatment consists of high-frequency (10 Hz) rTMS delivered to the
left dorsolateral prefrontal cortex at 110% resting motor threshold, with intensity
adjusted for scalp-to-cortex distance. The stimulation target is individualized using
task-based fMRI activation from a Tower of London planning task and neuronavigation
(Localite). Sham sessions are performed with a placebo coil, that mimics sound and
sensation, and at 60% motor threshold, so that it delivers no effective magnetic field.
Participants and researchers involved in clinical assessments, data collection, and
analysis remain blinded to allocation; technicians are trained to avoid any disclosure
that could compromise masking. To enhance feasibility for participants with fatigue and
sensory sensitivity, the treatment environment is kept low-stimulus with reduced lighting
and noise, scheduling is flexible with a weekly buffer option, and the minimum 16-session
option supports completion.

Endpoints and analysis The primary endpoint is change in fatigue from baseline to
post-treatment, analyzed as the between-group difference in pre- to post-treatment change
under the intention-to-treat principle. Secondary endpoints span cognitive functioning,
mood, sleep, quality of life, and physical performance, together with multimodal
neuroimaging and blood-based biomarkers that analyse neuronal integrity, cerebral blood
flow, functional connectivity, and inflammation. Statistical analyses use linear
mixed-effects models with appropriate covariates and multiplicity is handled via
Bonferroni for secondary endpoints and FDR for neuroimaging and exploratory analyses.

Safety and ethics Safety is monitored throughout treatment and follow-up via standardized
AE questionnaires and weekly tolerability questions. Known contraindications to rTMS/MRI
are applied at screening, and procedures for managing common side effects and rare events
are in place. The study is conducted per the Declaration of Helsinki and national
regulations, with approval by the METc Amsterdam UMC. Participants provide written
informed consent, travel costs are reimbursed, and a small participation compensation is
provided.

Recruitment and setting Recruitment is coordinated through the Post Covid Network
Netherlands patient portal, enabling efficient identification of potentially eligible
individuals who consented to be approached for research. The trial is conducted at
Amsterdam UMC.

Conclusion If the intervention proves effective, this study will provide the first
placebo-controlled evidence for high-frequency rTMS to reduce fatigue in long COVID, with
neuroimaging and blood biomarkers to shed light on underlying disease and treatment
mechanisms. Even if no between-group difference is observed, the trial will yield
valuable information on PASC and underlying mechanisms.