Operant H-reflex Down-conditioning of Rectus Femoris in Post-stroke Stiff Knee Gait
Phase: EARLY_PHASE1
Conditions: Training
Sponsor: University of Texas at Austin

 
 
 
 
 
 
 
 
 
 
 
 
 
Study Title: Operant H-reflex Down-conditioning of Rectus Femoris in Post-stroke Stiff Knee 
Gait 
 
 
NCT number: NCT05467774 
 
Document Date: 7/28/2021 
 
 

SUBJECTS 
A total of 7 participants (5 unimpaired [H1-H5, ages 20-26 years, 3 M/ 2 F], 2 post-stroke [S1-S2, 
1 M/1 F) were recruited for this study.  Each participant was able to stand for 10-minute intervals 
unassisted, walk on a treadmill for 10-minutes, and provide informed consent. Exclusion criteria 
included: lower limb musculoskeletal injury (e.g., fractures, sprains, strains, tendonitis, or bursitis), 
functionally relevant weight-bearing restrictions, vision impairment and must not have taken 
antispasmodic medication one day prior to the session. All 7 people participated in down-
conditioning. The study was approved by the University of Texas Institutional Review Board, and 
all subjects gave informed consent prior to participation. Both post-stroke individuals had right 
hemiparesis and the experiment was conducted on the right side.  
ACQUISITION AND PROCEDURES 
The experimental setup consisted of 8-channel surface electromyography (EMG) sensors (AMT-
8, Bortec, Calgary, AL), bipolar electrodes (Ag-AgCl, Noraxon, Scottsdale, AZ) a constant current 
electrical stimulator (Digitimer DS8R, Hertfordshire, UK), a data acquisition board (NI-PCIe 
6321, Austin, TX), a desktop computer, and EPOCS software (Fig 1). Additionally, during 
assessment sessions, stroke subjects walked on an instrumented split-belt force treadmill (Bertec, 
Columbus, OH), during which the ground reaction forces (GRF) were recorded at 1kHz, and lower 
limb kinematic data were recorded via inertial motion capture (IMU, Xsens, Enschede, 
Netherlands) at 60 Hz.  
 
The experiment was conducted over 30 experimental sessions per individual, with 3 sessions per 
week on non-consecutive days. To prevent diurnal variation in H-reflex magnitude, all training 

was performed at the same time of the day for each individual. Each experimental session consisted 
of preparation, femoral nerve navigation, recruitment and stimulation stages. The preparation 
stage started with drawing a grid on the surface of the femoral triangle on the leg. The femoral 
nerve was identified using an anatomical landmark-based method known as the “four-three finger 
technique”. Surface EMG electrodes were attached over the muscle belly of the rectus femoris 
(RF), medial hamstring (MH), vastus medialis (VM), and vastus lateralis (VL) based on commonly 
used anatomical landmarks31,32. RF was the primary focus for H-reflex monitoring, MH was 
monitored for antagonist activity, and VM and VL were monitored to validate the training 
specificity of RF H-reflex conditioning. EMG activity was amplified, bandpass filtered (3-1,000 
Hz), and sampled at 1 kHz. 
 
 
Fig 1 Experimental setup and visual feedback of RF (rectus femoris) operant H-reflex 
conditioning. (a) Participant with electrodes on the right leg is in the upright standing posture, 
facing visual feedback from a computer monitor. (b) During control/baseline trials, only the 

background EMG level of the RF was provided on the left bar graph. If the background EMG level 
of the RF and its antagonist pair MH (medial hamstrings) was within the shaded region, the bar 
remained green and the electrical stimulation was delivered. Otherwise, the bar turned red with no 
stimulation or trial progression. (c) During the training trial, RF H-reflex size and cumulative 
success rate were provided along with background EMG. The bar turned green if the RF H-reflex 
was below the threshold and turned red if larger than the threshold. 
 
During the femoral nerve navigation stage, the optimal stimulation location of the femoral 
nerve was established via a monopolar probe electrode to each point on the grid drawn on the 
femoral triangle34. Electrical stimulation was delivered as a square stimulus pulse, 1 ms in duration, 
intensity ranging between 10-20 mA or above depending on the individual, to elicit a reflex 
response with the subject in quiet standing. We monitored the time-course of the reflex response. 
The H-reflex was determined as the peak response following the initial stimulus artifact (<3 ms) 
and M-wave (~10-25 ms), typically within 30-40 ms post-stimulus. We used the following criteria 
to identify the optimal H-reflex response: 1) a clear biphasic shape, 2) a clear distinction from the 
M-wave or in case of contamination the biphasic signal lies on the downward slope of the M-wave, 
and 3) largest signal magnitude of all the candidate spots. After the optimal stimulation spot was 
located, the monopolar probe was replaced with a disposable Ag/AgCl snap electrode (circular, 
1cm diameter) and the anode electrode (square 5x5cm) was placed over the surface of gluteus 
maximus on the ipsilateral side. To avoid session-to-session variability in electrode locations, the 
positions of all electrodes were marked using semi-permanent surgical skin marker. After 
electrodes were placed, a maximum voluntary contraction (MVC) for RF and MH were measured 
separately. Participants were asked to sit on a chair with their ankle fastened with a belt to the leg 

of the chair and knee flexed to 90°. The investigator asked the participant to extend or flex their 
knee with maximum effort for 5-7 s. The participant repeated this three times with 15 s intervals 
in between. The representative MVC was determined as the mean of three MVC trials. 
During the recruitment stage, the RF H-reflex recruitment curve was achieved with the 
subject in a quiet standing posture. Electrical stimulation was delivered at 0.14 Hz only if the 
background EMG level of RF and MH were within a 10-20% MVC window respectively, 
representing a slight tonic voluntary activation to avoid altering reflex excitability. To find the 
maximum M-wave amplitude of the RF, we increased stimulation intensity by 2 mA increments 
starting at 10 mA until the RF M-wave amplitude remained constant despite increasing current in 
three successive stimulus intensities, defined as Mmax. For each stimulus intensity, two H-reflex 
responses were recorded. The H-reflex size was calculated as the mean of the peak-to-peak 
amplitudes of the two responses. The corresponding intensity (Imax) that elicited maximal 
representative H-reflex (Hmax) was used in the following trials. Post-stroke individuals did not 
require any additional support for standing, however for safety purposes a handrail was placed in 
front of the individual. 
During the stimulation stage, also in performed in a standing posture, the femoral nerve 
was stimulated at Imax at 0.14 Hz based on the same 10-20% MVC requirement. The first 6 sessions 
were baseline sessions with no feedback of RF H-reflex. Each baseline session was composed of 
3 runs of 75 trials, where each trial was a single stimulation pulse. The next 24 sessions were 
training sessions. During training, each session began with 20 control trials with no feedback, 
followed by 3 runs of 75 trials using the peak-to-peak magnitude of RF H-reflex as visual feedback 
to the participant (Fig 1). During training sessions trials, M-wave size was monitored, and stimulus 
strength (Imax) was minimally adjusted to maintain the predetermined M-wave size. Post-stroke 

individuals participated in an additional 2 assessment sessions, one before training and one after 
training sessions. In participant H4, in two of the 30 training sessions, the H-reflex showed overlap 
with the M-wave. In offline analysis, we used a best decaying exponential fit to obtain the H-reflex 
estimate as illustrated in our earlier work.   
FEEDBACK 
As shown in Fig 1c, visual feedback consisted of 1) RF and MH background EMG (left 
bar), 2) RF H-reflex (right bar), 3) trial number, and 4) cumulative success rate. The left bar was 
green if the background EMG was below activation threshold but was red otherwise. The H-reflex 
stimulation was delivered only when the background EMG bar was green. The participant was 
provided with the objective to reduce the height of the right bar, representing H-reflex amplitude, 
below a given threshold (shaded area in Fig 1c). The threshold was updated for each run as the 
66th percentile of the H-reflex trials from the previous run. If below the performance threshold, the 
right bar turned green and cumulative success rate was adjusted accordingly. If above the 
threshold, the bar turned red, and the success rate fell. Each trial was 7 s duration, with a single 
stimulation pulse stimulation of 1 ms pulse width followed by feedback for the remainder of the 
trial. Each session was recorded on a different non-consecutive day, up to 3 times per week. The 
6 baseline sessions each consisted of 3 runs of 75 control trials (225 total), while each of 24 training 
sessions consisted of 20 control trials and 3 runs of 75 training trials (245 total) in standing posture. 
Subjects were exposed to H-reflex feedback only during the training trials, and no H-reflex 
feedback was given during the control trials. The participant’s score for each run was cumulative 
success rate (𝑆𝑆𝑆𝑆𝑆𝑆=
𝑁𝑁𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠
𝑁𝑁𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 %) and participants were rewarded in proportion to one’s score (i.e., 
𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅($) = 1.0 + 0.05 ∗(𝑆𝑆𝑆𝑆𝑆𝑆−50)). If the participant was successful in achieving three 

consecutive success rates that were equal to or higher than 90% for all three runs, they were 
provided with an extra dollar as bonus, which made the maximum additional compensation amount 
$10.00. A rest period was provided between each run to minimize fatigue, which could affect 
muscular responses to stimuli. 
CLINICAL ASSESSMENTS 
The two individuals with stroke participated in pre and post training assessment sessions. 
Participants performed a 10 m walk test (10MWT) with instructions to walk with maximum effort. 
This process was repeated 4 times and the participant’s gait kinematics were recorded using inertial 
motion capture. Next, participants completed the quadriceps pendulum test, which is a non-
invasive biomechanical method of evaluating spasticity using gravity to provoke muscle stretch 
reflexes during passive swinging of the lower limb. Inertial measurement units were attached on 
top of thigh, shank, and foot to calculate the knee joint angle during the process. This test was 
repeated 3 times with 30 second rest periods in between. The main outcome of this test was 
quadriceps reflex threshold angle (QRTA), which was the angle difference from the maximum 
knee extension position to the first swing excursion, the first transition period of knee flexion to 
knee extension. Lastly, the participant was asked to walk on an instrumented treadmill while their 
gait kinematics, ground reaction forces (GRF), and H-reflexes were obtained. During treadmill 
walking, four gait phases (heel-strike, toe-off, mid-stance, and mid-swing) were detected in real-
time using online GRF data using conventional gait analysis1. Heel-strike was detected when GRF 
value exceeded a predetermined force threshold (Fth) for the first time (0% gait cycle) and toe-off 
was detected with GRF dropping below the threshold (Fth) (60% gait cycle). Mid-stance was 
detected as the time point when 30% of step duration, pre-calculated from one’s step length and 
gait speed, had elapsed from the moment of heel-strike. Mid-swing was determined in a similar 

manner, which was detected at the 80% gait cycle. The H-reflex was elicited and measured only 
when a desired gait phase was detected and occurred at least 7 s after the last H-reflex trial. Twenty 
H-reflexes were recorded for each gait phase. A 5 min break after each of four runs was provided 
to minimize the effect of fatigue on H-reflex. The main outcomes consisted of 1) knee flexion 
range of motion (RoM), 2) peak knee flexion velocity, and 3) H-reflex amplitude during the four 
different gait phases. 
OUTCOME MEASURES 
We normalized the H-reflex measurements. For each session, the H-reflex was normalized with 
the session’s maximum motor response (M-wave, Mmax), which is the most commonly used 
method of normalization40. To directly compare between session-to-session performances, each 
session’s H-reflex trial was averaged, normalized by Mmax, and represented as a percentage (%) of 
the mean H-reflex of 6 baseline sessions. Main outcome measures of each session were the average 
of 225 peak-to-peak H-reflex responses from the RF and VM.  
STATISTICAL ANALYSIS 
We investigated the hypothesis that RF H-reflex can be down-conditioned through operant H-
reflex conditioning. We first verified normality of the data using a Shapiro-Wilk test. To validate 
the effect of training, a paired t-test (a<0.05) was used to compare the mean of the 225 normalized 
H-reflex trials during 6 baseline sessions to that of the last 6 training sessions.  
 

ETHICS APPROVAL  
This study was conducted according to the Declaration of Helsinki and had ethical approval from 
the University of Texas Institutional Review Board. All subjects gave written informed consent 
prior to data collection.