The Effects of Exercise Training on Central and Peripheral Blood Flow Regulation in Individuals With Down Syndrome
Phase: NA
Conditions: Down Syndrome
Sponsor: University of Nevada, Las Vegas

Study Protocol Effects of Exercise on Blood Flow Regulation in Adults 
with Down Syndrome (NCT04854122, approved 03/02/2022) 
 
Contents 
Aims .......................................................................................................................................................... 2 
Lay language summary ............................................................................................................................. 2 
Protocol and procedures .......................................................................................................................... 2 
Design .................................................................................................................................................... 2 
Description of study procedures ........................................................................................................... 2 
Familiarization sessions. ................................................................................................................... 2 
Description data collection visits. ..................................................................................................... 3 
The first visit ...................................................................................................................................... 3 
Measurements ...................................................................................................................................... 4 
Visit 1 for all participants .................................................................................................................. 4 
Baseline measures ........................................................................................................................ 4 
Visit 2 for individuals with Down syndrome (added to visit 1 for individuals without Down 
syndrome) ......................................................................................................................................... 5 
Graded exercise test treadmill ...................................................................................................... 5 
Procedures ................................................................................................................................ 5 
Measurements .......................................................................................................................... 5 
Gait and balance test protocol ...................................................................................................... 5 
Visit 3 for individuals with Down syndrome (visit 2 for individuals without Down syndrome) ....... 6 
Hand grip exercise protocol .......................................................................................................... 6 
Procedures ................................................................................................................................ 6 
Measurements hand grip exercise protocol ............................................................................. 7 
Statistical Analysis ..................................................................................................................................... 8 
 
 
 
 

Aims 
To investigate the chronic response to exercise by conducting an exercise intervention in individuals 
with Down syndrome, to determine to what extent the improvements in work capacity will be explained 
by changes in central or peripheral blood flow regulation. 
  
Aim 1: Determine if exercise training improves peripheral or central regulation of blood flow in 
individuals with Down syndrome compared to individuals with Down syndrome in a control group and to 
individuals without DS. 
Aim 2: Determine if exercise training improves the cardiovascular autonomic profile in individuals with 
Down syndrome compared to individuals with Down syndrome in a control group and to individuals 
without DS. 
Aim 3: Determine if exercise training improves gait and balance in individuals with Down syndrome 
compared to individuals with Down syndrome in a control group and to individuals without DS. 
 
Lay language summary 
Work capacity is an important predictor of declining health or physical function, and of mortality, and is 
commonly measured as peak oxygen consumption. Peak oxygen consumption is very low in individuals 
with Down syndrome, the most prevalent genetic cause of intellectual disability. Previous research 
suggests individuals with Down syndrome may experience a double disadvantage when they are 
exercising: they may not be able to increase cardiac output sufficiently and they may not be able to 
allocate adequate blood flow to the working muscles. The aim of this research proposal is therefore to 
investigate the responses in central and peripheral blood flow regulation and cardiac autonomic 
function to exercise training in individuals with DS. Additionally we investigate the effects of exercise on 
gait, balance, level of support and quality of life in individuals with DS. 
 
Protocol and procedures 
 
Design  
 
This intervention study will be set up as a randomized controlled trial comparing the effects of a 12-
weeks exercise program to 12-weeks of non-exercise usual care in participants with Down syndrome 
and to a control group of individuals without Down syndrome. 
 
Description of study procedures 
 
Familiarization sessions.  
We involve parents/care givers in order to provide a supportive environment for participants and to 
enhance the parent/caregiver understanding of the research. Prior to study initiation, photographs and 
video clips of the laboratory and study equipment will be provided to help the participants become 
comfortable with the laboratory environment and equipment, and the informed consent information is 
sent to the participants and their parents/care-givers for their review. Familiarization of the data 
collection procedures for the participants with Down syndrome will be divided in two parts: they will 
practice and become accustomed with the treadmill and the equipment for the graded maximal exercise 

test (first visit), and they will practice and become accustomed with the procedures for the hand grip 
exercise protocol and the LBNP (second visit).  
 
Description data collection visits.  
After discussing the study and potential questions either over the phone or in person, participants 
(and/or parents/caregivers for the group with Down syndrome) will complete a health history 
questionnaire and physical activity questionnaire to confirm eligibility. The Health History Questionnaire 
includes all the component of the new ACSM guidelines for the Preparticipation Health Screening for 
Exercise (Riebe et al. 2014, MSSE). Eligible participants with Down syndrome and their parent/care-giver 
will then provide written informed consent, or give verbal assent (for individuals with Down syndrome), 
and the eligible control participants will provide informed consent themselves. Participants are 
randomized directly after we have received their informed consent.  
 
The first visit 
For women of childbearing age, the first visit will include a urine pregnancy test using a test stick. 
Females will be studied during the first 3-5 days of menses or during the placebo phase if taking oral 
contraceptives, in order to control for hormonal variation. For participants with Down syndrome, the 
first visit continues with baseline measures (see below) and familiarization with the graded maximal 
exercise test protocol. If necessary, additional familiarization sessions will be scheduled for people with 
Down syndrome.  
 
The second visit  
We perform the graded maximal exercise test, the gait and balance test protocol, and familiarize the 
participant with the procedures for the third visit: the hand grip exercise protocol and the LBNP. The 
third visit we assess the peripheral blood flow during the hand grip exercise protocol without and with 
the LBNP (randomized order) (see figure below). At the end of the third visit, we will randomize the 
participants with Down syndrome to the exercise or the usual-care group. The participants randomized 
to the usual-care group will have the option to participate in the exercise program after completing their 
measurements at the end of the 12 weeks of usual care. For control subjects without Down syndrome, 
the first and second visit are combined. Their second visit is the same as the third visit for individuals 
with Down syndrome, however, they will not continue with any intervention, as their participation only 
includes baseline measurements. 
 
 
 
Third visit lab 
Grip strength and 
LBNP protocol 
48 hrs between 
E-Mail/ telephone: 
Screening for 
eligibility 
Second visit lab 
Familiarization grip 
strength/LBNP + 
Actual max test 
Time 
First visit lab 
Informed 
consent 
Baseline measures+ 
familiarization 
treadmill test 

Intervention. For the individuals with Down syndrome who are randomized to the exercise intervention: 
the exercise intervention will last 12 weeks and will consist of a supervised combined aerobic and 
resistance training program with a frequency of 3 days/week, and use a program comparable to 
Mendonca et al.[1, 2]. The program we will be using is specifically developed for individuals with Down 
syndrome based on the Mann Method PT Principles. The MMPT Principles focus on a progressive 
program of therapeutic activity (cardiovascular activity), therapeutic exercise (foundational 
strengthening and hip strengthening activities), and neuromuscular rehabilitation (visual/vestibular and 
balance activities) to address the unique needs of individuals with Down syndrome. This program has 
been developed and successfully implemented in Down syndrome activity centers across the country. 
The trainer will discuss with the participant what days/times work best for them. The exercise sessions 
will be offered as a remote exercise program, which means that all sessions will take place online 
through a HIPAA compliant Zoom platform. The trainer is part of the research team and will be a 
certified PT or a Personal Trainer certification with at least a bachelor degree in Exercise Science of 
Kinesiology. In the exercise sessions, the participant will work on their strength, balance and aerobic 
endurance. Each session consists of 10 min of strength exercises (Foundational Exercise), 10 min of Hip 
Strengthening, 10 min of Vestibular and Balance Exercise, and 20 min of Aerobic Exercise, and starts 
with a warming up and ends with stretching/cooling down. Each new exercise will be introduced in easy 
steps and practiced until the participant is comfortable executing it. After a 5 min. warm-up, the 
participant will perform 20 min of aerobic exercise at a heart rate of 65% of your maximum heart rate, 
which increases to 65-85% during the later weeks of the program. The resistance exercise part will 
include all major muscle groups, both upper and lower body, using multi- and single-joint exercises. 
Balance training will include exercises such as single leg stance and compliant surfaces with eyes open 
and closed, tandem stance with eyes open and closed, dual task walking, walking with change in speed, 
head turns, stepping over obstacles, external perturbations with eyes opened and closed, medicine ball 
toss. The caregiver does not need to be present during the exercise sessions. 
The participants randomized to the control condition will continue with their usual daily routines for 12 
weeks and then follow the same post-intervention data collection protocol as the intervention group, 
which is identical to the baseline collection. Participants in the control condition will be offered the 
opportunity to participate in the online exercise program after the post-intervention data collection, but 
this will fall outside the scope of this study.  
 
Measurements 
 
Work capacity, as measured by peak oxygen uptake with a graded exercise test on a treadmill, is the 
primary outcome variable. For the participants with Down syndrome, all measurements will be carried 
out pre- and post training under the same conditions and at the same time of day, for the control group 
without Down syndrome all measurements will be carried out as baseline measurements only. 
 
Visit 1 for all participants 
 
Baseline measures 
Participants will be tested in a postprandial state (>3 h) on 2 separate days and will refrain from exercise 
24 h before each test day. Participants will be also asked to refrain from drinking or eating caffeine and 
drinking alcohol on testing days. Participants who do not follow the requirements on study days will be 

asked to come back another day. During the first visit, the participants will complete questionnaires 
about risk of falling (Short Falls Efficacy Scale-1), perform a cognitive function test on a laptop 
specifically designed for individuals with Down syndrome evaluating the following domains: 
Multitasking, Episodic memory, Executive function and Processing speed (Cambridge 
Neuropsychological Test Automated Battery (CANTAB)). After that, height, weight and circumferences 
will be measured. Body composition will be determined with a DEXA scan, to determine forearm 
composition and mass. Vascular health (arterial stiffness and central blood pressure) will be measured 
with the Complior Analyse device. Two-dimensional echocardiography (Hitachi Arietta 70 system, Tokyo, 
Japan) will be used to measure aortic root diameter, cardiac output, and stroke volume and end systolic 
volume at rest. In addition, clinical autonomic function tests (heart rate variability at rest and recovery 
after exercise, deep breathing, Valsalva maneuver, isometric exercise, active and passive orthostasis) 
will be evaluated during the first study visit. At the end of the first visit, all participants will be provided 
with an Actigraph and instructions to wear this device for 7 days.  
 
Visit 2 for individuals with Down syndrome (added to visit 1 for individuals without Down syndrome) 
 
Graded exercise test treadmill 
 
Procedures 
The participants will be familiarized with the graded maximal test protocol on the treadmill. The graded 
maximal exercise test begins with a submaximal horizontal walk on a motorized treadmill at a 
individualized comfortable speed, using a validated and reliable protocol for this population [3, 4]. Grade 
will be increased 2.5% every 2 min until a 12.5% grade will be reached. From this point, grade will be 
held constant whereas speed will be increased by 0.5 mph every minute until exhaustion. This may 
involve running at the later stages. The exercise test will be terminated when the participant can no 
longer keep up with the treadmill speed or shows signs of volitional fatigue. Heart rate, blood pressure 
and oxygen uptake (VO2) will be monitored continuously throughout the exercise and recovery [5]. The 
highest VO2 will be recorded as VO2peak if the respiratory exchange ratio is over 1.0 [6].  
 
Measurements 
Cardiac output will be measured using ultrasonography, by measuring the aortic diameter at the level of 
the valve form the peristernal long axis. Ascending aortic blood flow will be measured using continuous 
Doppler echocardiography using a pedoff probe in the suprasternal notch as we have previously 
described [7]. 
 
Gait and balance test protocol 
 
This is a 30-min protocol evaluating different aspects of gait and balance with valid and reliable, easy, 
participant-friendly, quick and functional tests. For the Foot Posture Index the participant stands and the 
test instructor evaluates different aspects. This will be evaluated with and without the supportive 
footwear. The Timed Up and Go test is a timed test where the participant stands up from a chair, walk 
10ft, turn around, walk back and sit down again, as fast as possible. For the Stair Climb test the 
participant is timed when climbing up a flight of 9 stairs. After that the participant performs a Functional 
Reach Test in the Virtubalance system, which electronically records how far the participant can reach 

forward.  After that the participant will be timed walking over a 30ft distance at a comfortable pace to 
determine Gait Speed (average of 3 attempts), and then will be walking that distance at his/her fastest 
walking pace, to determine Fast Walking Speed (average of 3 attempts). The participants will be 
executing these walking bouts on the GaitMat to evaluate spatial and temporal gait parameters and 
they will be recorded with 2D camera to determine lower extremity kinematics (knee and hip flexion, 
knee valgus/varus). To conclude, the participant will perform several standing balance tasks (Bertec 
Balance Measures) on a force platform. We will use a harness for safety, and the participant will be 
asked to lean in different directions following a target on a screen (evaluating the limits of stability), to 
maintain position while the platform moves in different directions (evaluating motor control) and to 
stand still for 3x 20 seconds with eyes open and 3x 20 seconds with eyes closed (evaluation sensory 
organization).  
 
Visit 3 for individuals with Down syndrome (visit 2 for individuals without Down syndrome) 
 
Hand grip exercise protocol 
 
Procedures 
Participants will be placed in a supine position with his/her lower body in the LBNP chamber, sealed at 
the waist. They will be instrumented with a 3 lead ECG to measure heart rate continuously and beat-to-
beat blood pressure will be measured using finger plethysmography (Finapres NOVA, TNO Biomedical 
Instrumentation, The Netherlands). 
Resting measurements will be conducted following a 10 min supine rest period. After the resting 
measurements, the participants are asked to squeeze the hand grip dynamometer with maximal force. 
This is repeated three times in total, with 1 minute of recovery in between. The highest is used as the 
participant’s maximal voluntary contraction (MVC). Then the different parts of the hand grip exercise 
protocols start, with randomization of the order of Part 2B and 2C. 
Part 2A:  Diameters, blood flow and vascular conductance in the brachial artery will be measured in 
response to a short, single contraction of hand grip exercise with a hand grip dynamometer of 1 sec, at 
15% and 30% of the participant’s MVC. The peak blood flow within the first 4-6 cardiac cycles following 
the contraction will be used to determine the immediate contraction-induced vasodilation. These 
measures will be performed in triplicate, with 1 minutes of rest in between. 
Part 2B: The vasodilatory response to dynamic hand grip exercise (2 sec rest, 1 sec squeeze for 4 
minutes) at 15% and 30% of the participant’s MVC (with 5 min of rest in between the different 
intensities) will be investigated. The change in diameters and blood flow of the brachial artery will be 
measured in the exercising arm with Doppler ultrasound in the last 30 sec of each condition. 
Part 2C: The ability to vasodilate will be further challenged by adding a mild lower body negative 
pressure (LBNP) of -20 mmHg and performing the same dynamic hand grip exercise protocol and 
measurements as described in Part 2B. All measurements will be repeated during the last 30 seconds of 
the LBNP application. LBNP will be stopped if systolic blood pressure drops below 80 mmHg or if 
participants exhibit symptoms of pre-syncope. However, this protocol has been well tolerated in our 
pilot studies for participants both with and without Down syndrome. 
 
 
 

Measurements hand grip exercise protocol  
 
Forearm blood flow and vascular conductance will be measured in both the exercising and non-
exercising arm using high definition ultrasound (Arietta 70, Aloka-Hitachi). The brachial artery will be 
imaged in dual mode allowing for simultaneous determination of artery diameter (B-mode) and flow 
velocity (Doppler mode). Blood flow will be determined from the following formula: Forearm Blood flow 
= (Mean blood velocity) x (Brachial Cross Sectional Area) x (60) and expressed as ml/min. Forearm 
vascular conductance will be determined by dividing forearm blood flow by mean arterial pressure. 
Forearm blood flow and vascular conductance will be normalized to forearm lean mass to account for 
differences in lean mass between individuals.  
 
Muscle oxygenation and capillary blood flow will be measured with near-infrared spectography (NIRS), 
which is a simple, noninvasive method for measuring the presence of oxygen in muscle [8]. It can 
monitor changes in muscle oxygenation and blood flow during submaximal and maximal exercise. 
During exercise, the extent to which skeletal muscles deoxygenate varies according to the type of 
muscle, type of exercise and blood flow response[9]. Pulmonary oxygen consumption will be measured 
using an open-circuit breath-by-breath system (BIOPAC). Capillary blood flow can then be estimated by 
rearranging the Fick equation to solve for bloodflow (Q):  Qcapillary = VO2muscle/ (arterio – venous 
O2difference), using the pulmonary oxygen uptake (VO2 ) and deoxyhaemoglobin (HHb) derived from 
NIRS to represent VO2muscle and (a −v)O2, respectively[10, 11]. 
 
Venous blood samples will be obtained from the participants with and without Down syndrome before 
and at the end of 5 min LBNP without hand grip exercise. A trained and licensed phlebotomist will 
obtain a total of 70 mL (60 mL before LBNP (approximately 4.5 tablespoons), 10 mL at the end of 5 min 
LBNP (approximately 0.75 tablespoons) blood sample via sterile venipuncture from the antecubital vein. 
The plasma will be analyzed for catecholamine concentrations (epinephrine and norepinephrine) with 
enzyme-linked immuno sorbent assays (ELISA kit). Additionally, the baseline samples pre- and post will 
be analyzed for C-reactive protein, TNF-alpha and interleukin-6 for inflammatory; insulin, glucose, leptin, 
adiponectin and lipid profile; and thyroid stimulating hormone as a marker for thyroid function. Based 
on previous research in Down syndrome on antioxidants, we will include superoxide dismutase-1 (SOD-
1), catalase (CAT) and glutathione peroxidase-1 (GPX-1) [12, 13]. The generation of reactive oxygen 
species is increased in DS because of the mitochondrial dysfunction [14], but as the reactive oxygen 
species react too quickly to measure, the resulting oxidative products are used as proxy measures. We 
therefore include lipid peroxidation (measured by malondialhyde (MDA)), myeloperoxide (MPO, 
catalyzes the conversion of hydrogen peroxide to other reactive oxygen species), and oxidized low 
density lipoprotein (OxLDL). These oxidative products are all measurable in plasma or serum, and we will 
follow the specified ELISA procedures where applicable. We will also include a large-scale gene 
expression analyses for mitochondrial dysfunction and oxidative stress, which are located on 
chromosome 21, the chromosome affected by the trisomy 21 characteristic for individuals with DS. To 
this end, NextGen mRNA seq will be used to analyze the expression of SOD-1 and APP and a broad 
spectrum of expression pathways that are altered in the DS state. As an option, subjects may provide 
consent to bank any leftover blood samples upon study completion for use in future research about 
cardiovascular disease.  The samples will be stored de-identified in the UNLV Translational Biomarker 
Unit. 

Statistical Analysis 
Descriptive statistics will be used to present baseline characteristics. A independent t-test will be 
used to test for differences in the change from pre to post between the exercise group and the 
control group. Additionally, a two-way mixed ANOVA will be used to evaluate condition (exercise 
and control) by time (pre-post) effects of the exercise program on the outcome measures. The a 
priori alpha level is set at 0.05.