Walking fatigability is a motor impairment happening in approximately 43.5% of people with multiple sclerosis (pwMS). Walking fatigability is defined as a decrease in 10% of distance walked, comparing the first minute with the last minute of the 6-minute walking test (6MWT), which one may call distance walking fatigability. More recently, our research group has found that walking fatigability manifests also in terms of gait quality, with different gait characteristics affected from patient to patient, suggesting that different gait profiles might be observed for walking fatigability. One possible explanation for the walking fatigability motor impairment is the lower capacity or neural reserve of pwMS to sustain optimal levels of cortical activity to either maintain performance (i.e., distance walked) or gait quality.

The study proposes two research questions. Both part A and B are observational studies.

The study includes 70 pwMS and 30 healthy controls. The study consists of 3 tests. The clinical assessment session will be composed of responses to different questionnaires, while parts A and B comprehend walking overground (part A) or on fixed-speed treadmill (part B).

Part A Session 1 (1 hour and 30minutes, including equipment preparation and rest time).

First, anthropometric characteristics and measurements of force, spasticity, cognition, walking speed, and performance foot tap test will be performed. For cortical activity measurement of the prefrontal cortex, supplementary motor area, premotor cortex, and primary motor cortex, a functional Nears-infrared Spectroscopy (fNIRS) will be positioned in the participant's head. Wearable sensors will be positioned in a pre-defined body position for the measurement of gait characteristics. Both gait characteristics and cortical brain activation will be measured concomitantly during fast and comfortable speed short walking (10 trials of 15 seconds with 25-30 seconds standing still resting periods between trials, in a block design) before and after and during a prolonged walking (6-minute walking test (6MWT)) protocols. A resting period of 20 minutes will be provided to the participants before the beginning of the 6MWT.

Part B. Participants who participated in Part A will be invited to participate in Part B. This study also consists of a single visit that is 1 hour and 30 minutes long (preparation and assessment included). The participants will be asked to walk on an immersive treadmill in two fixed-speed conditions (relative to 80% and 50% of the Timed 25-foot Walking test) while infrared cameras will record the reflective markers position. Forty minutes of rest will be provided to the participants between the fixed-speed conditions.

Methodology Recruitment strategy. Participants will be recruited from the Flemish MS rehabilitation centers Melsbroek (NMSC) and Overpelt (Noorderhart RMSC), as well as the REVAL research center inUHasselt. Information flyers will be distributed in the centers and via information placed on the website and social media of REVAL and the Flemish MS society. We will search age and sex-matching subjects for the healthy controls, considering a 5-year range per subject.

After giving informed consent, demographics of age and gender, as well as MS-related information such as EDSS, type of MS, and years since diagnosis, will be collected and provided by the centers or via self-report. The participants will be instructed to keep their normal routine and avoid extraneous physical activity at least 24 hours before the data collection session.

Experiment outline. The study proposes two research questions and will be performed in three days. The first day is the clinical assessment/screening of the participants (common for Part A and B). Part A is an observational study (2nd day), and Part B is an observational and feasibility study (3rd day).

Clinical assessment/screening The participants will be contacted to verify their interest and eligibility (inclusion and exclusion criteria) to participate in the study. In the case of inclusion in the study, different questionnaires will be sent to the participants to fill in: Falls Efficacy Scale International (FES-I) for fear of falling, Multiple Sclerosis Walking Scales (MSWS-12) and 10 questions encompassing gait items for walking perceived ability [11], Pittsburgh Fatigability Scale (PFS) for perceived fatigability, Modified Fatigue Impact Scale (MFIS) for perceived fatigue, the Brief Pain Inventory (BPI, short form) for the assessment of perceived pain, Multiple Sclerosis Spasticity Scale 88 (MSWS-88, section 5 and 6) to verify the impact of spasticity in the walking and movement ability and Brief Self-Control Scale (BSCS) for self-control evaluation. Specifically for the pwMS, the Patients Disease Determined Steps (PDDS) will be asked in order to know the self-perceived level of disability. Participants will also be asked and oriented to measure their head's circumference for the functional near-infrared spectroscopy (fNIRS) measurement. In case the patient is contacted in the MS centers (MS Rehabilitation Center- Pelt and National MS Center Melsbroek), the therapist will provide the questionnaires, measure the head size of the participants, and obtain the level of disability (measured by the expanded disability status scale- EDSS). The skin color, type, and color of the hair will be noted to assist in the fNIRS analysis.

Part A- Assessment The study includes 70 pwMS and 30 healthy controls. The study consists of a single visit (1 hour and 30 minutes duration, including equipment preparation and rest periods). Participants will be asked to wear shorts or legging pants for better fixation of the sensors. Anthropometric characteristics such as age, weight, height, and sex will be asked and measured. Moreover, for pwMS, the time from the diagnosis, MS phenotype, and medications that could influence fatigue or walking performance will also be recorded. The symbol digit modalities test (SDMT) will evaluate the cognitive processing speed. Afterward, the modified Ashworth scale (MAS) will be used to check the muscle spasticity of the knee flexors/extensors and ankle flexors/extensors. The motricity index will be performed for muscle strength measurement, followed by the performance of the timed 25-foot walking test (T25FWT). For cortical activity measurement of the prefrontal cortex, supplementary motor area, premotor cortex, and primary motor cortex, a fNIRS will be positioned in the participants head. Wearable sensors will be positioned in a pre-defined body position to measure gait characteristics. Both gait characteristics and cortical brain activation will be measured concomitantly during short walking (10 trials of 15 seconds with 25-30 seconds standing still resting periods between trials, in a block design) before and after and during a prolonged walking (6-minute walking test (6MWT)) protocols. Before the beginning of the 6MWT, two questions will be asked to the participants: "Do you slow down when walking for a while?" and "Do you feel a worsening of your smoothness when walking for a while?" where they will have to rate between 1 ("not at all") and 5 ("Extremely"). A questionnaire about their perceived gait quality changes, comparing the end with the beginning of the 6MWT, will be asked through the Perception of Gait Quality Change (PGQC).

Part B- Assessment The study includes 30 pwMS and 30 healthy controls. Participants who participated in Part A will be invited to participate in Part B. Participants will be asked to wear shorts or legging pants for better fixation of the sensors and the reflective markers. This study also consists of a single visit of 1 hour and 30 minutes (preparation and assessment included). The wearable sensors will be positioned in the participants following the same setup as part A. Reflective markers will be positioned in body landmarks. The participants will be asked to walk on an immersive treadmill in two fixed-speed conditions (relative to 80% and 50% of the T25FW), while infrared cameras will record the reflective markers position. The participants will also be asked about previous experience walking on a treadmill.

PART A. ASSESSMENT. Outcome measures specifications Foot tap test. The participants will have to perform a foot tap test as fast and with higher amplitude as possible for 6 times: i) 2 times 10 seconds for each foot, with resting period of 1 minute between the trials and ii) one time, each foot, for 1 minute. The two times 10 seconds for each foot will be repeated after the short walking test after the 6MWT. Inertial measurement units will be position on the top of their feet for frequency and amplitude of the movement measurement concomitantly with the record of cortical brain activity measurement. A baseline period of 15 seconds, preceding each trial, will be performed for the cortical brain activity.

Short walking test. The participants will be instructed to walk at their maximum (5 times) and comfortable speed (5 times) in blocks of 15 seconds, with rest periods ranging from 25 to 30 seconds before and after the 6MWT. The order of the first four trials will be consistent among participants (starting with 2 fast walking followed by 2 comfortable walking). The remaining of the trials will be randomized. The different resting periods were chosen to prevent the participants from anticipating the beginning of the trial, minimizing the effects of anticipation on cortical brain activation. During the resting periods, the participants will be instructed to perform a simple cognitive task of counting down starting from 50 to prevent mind wandering. The software Psychophy will be used to draw the experimental procedure and synchronize the wearable sensors and fNIRS equipment. Standardized audio instructions will be provided to the participants on what they will have to do and the countdown for the beginning of the task.

Walking capacity and fatigability test. Participants will perform the 6MWT. During the 6MWT, participants will be instructed to safely walk as fast as possible to cover a maximal individual distance for 6 min on a 25m walkway with turns at both ends. No verbal encouragement will be provided to the participants and every minute completed of the test will be informed by the research staff. Patients will be allowed to use an assistive device if needed, and no rest during the 6MWT will be allowed.

Measuring gait:

Participants will be equipped with 10 wearable sensors (OPAL V.2, APDM, USA, https://www.apdm.com/wearable-sensors/) to measure gait characteristics in different gait domains. Two sensors will be strapped on their feet, two on each tibia, two on each thigh, two on each wrist, one on their lumbar (L4), and one on the sternum.

Measuring cortical brain activity:

The measurement of cortical brain activity will follow the guidelines to assess gait and posture using fNIRS . Participants will wear a portable fNIRS cap with a backpack containing the fNIRS device on the back. The size of the cap will change in accordance with the head size of each participant. The fNIRS system is composed of 16 sensors and 16 detectors and will be positioned in the cap to measure the following regions of interest: the prefrontal cortex (Broadman area 9, 10, and 46), supplementary motor area (Broadman area 6), premotor cortex (Broadman area 6), and primary motor cortex (Broadman area 4). These areas correspond to the areas involved in the brain's human control for walking.

PART B. ASSESSMENT. Outcome measures specifications 6-minute walking test (6MWT) fixed speed. The participants will perform the 6MWT in a two-belt treadmill, with two force platforms embedded, in the Gait Real-Time interactive laboratory (GRAIL), in two fixed speed conditions: i) 80% of the walking speed obtained in the T25FW and ii) 50% of the walking speed obtained in the T25FW. The order of the fixed-speed treadmill conditions will be randomized and counterbalanced between groups. Forty minutes will be provided to the participants between the fixed-speed conditions. The GRAIL (Motekforce Link, Amsterdam, the Netherlands) will assess gait characteristics changes over the fixed-speed 6MWT. This system allows real-time recording of body movement in a high-resolution recording by means of a 3D motion capture system (10 cameras Vicon Motion System® with a data collection frequency set at 100 Hz). Twenty-two passive reflective markers (HBM lower body model) will be positioned on the bony landmarks of participants.

Inclusion Criteria (part A and B):

• age between 18 and 65 years old
• a diagnosis of MS (2017 revisions of the McDonalds criteria) with EDSS 1 up to 6.5 (part A) and with EDSS 1 up to 5.5 (part B) OR minimal punctuation of 2 in the MSWS-12 for one of the items: 7, 10, 11, and 12 (for pwMS)
• no relapses 1 month preceding the start of the study (for pwMS)
• ability to walk for 6 minutes without rest

Exclusion Criteria (Part A and B):

• Cognitive impairment hindering understanding of study instructions,
• Pregnancy
• Musculoskeletal disorders in the lower limbs that are not related to MS.
• PwMS needing an assistive device to walk can not be included in part B given safety reasons.

Participants will be recruited from the Flemish MS rehabilitation centers Melsbroek (NMSC) and Overpelt (Noorderhart RMSC), as well as the REVAL research center in UHasselt. Information flyers will be distributed in the centers and via information placed on the website and social media of REVAL and the Flemish MS society. We will search age and sex-matching subjects for the healthy controls, considering a 5-year range per subject.