Mental Health and Physical Activity 15 (2018) 153–163

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Mental Health and Physical Activity

journal homepage: www.elsevier.com/locate/menpa

Does manipulation of arterial shear stress enhance cerebrovascular function T

and cognition in the aging brain? Design, rationale and recruitment for the
Preventia randomised clinical trial
Daniel J. Greena,∗, Kay L. Coxa,b, Johanna C. Badcockc, Philip N. Ainslied, Carmela Pestelle,
Barbara A. Maslena, Nicola T. Lautenschlagerf,g,h
a
School of Human Sciences (Exercise and Sport Science), University of Western Australia, Perth, WA, Australia
b
School of Medicine, University of Western Australia, Perth, WA, Australia
c
Centre for Clinical Research in Neuropsychiatry, University of Western Australia, Perth, WA, Australia
d
School of Health and Exercise Sciences, The University of British, Columbia, Canada
e
School of Psychological Science, University of Western Australia, Perth, WA, Australia
f
Academic Unit for Psychiatry of Old Age, Department of Psychiatry, The University of Melbourne, Melbourne, VIC, Australia
g
North Western Mental Health, Melbourne Health, Melbourne, VIC, Australia
h
WA Centre for Health & Ageing, University of Western Australia, Perth, WA, Australia

ABSTRACT

Background: Cognitive and cerebrovascular declines are potentially life-chan-

ging conditions which are associated with a substantial healthcare burden worldwide. While pharmacological approaches for the prevention of cognitive decline have
been largely ineffective, exercise programs may be able to delay or prevent cognitive decline in those at risk. One putative mechanism for the beneficial impacts of
exercise on brain health involves improvement in cerebrovascular function. Exercise positively affects arterial structure and function in peripheral arteries and
microvessels, but the impacts on brain vascular function are largely unknown. Water immersion increases brain blood flow and shear stress, a mechanism that
improves artery health. We hypothesised that repeated water immersion may lead to adaptations which enhance cerebrovascular function and health in humans.
Methods/design: 72 sedentary older adults (50 years and older) with subjective memory complaints were randomly assigned to a 24-week water-based or land-based
walking program (designed to elicit differing stresses on blood vessels) or an education group. Measures of cerebral artery function and cognitive performance were
assessed before and after the intervention, and again at 48 weeks to test for persistence of any benefits. Other outcomes included biochemistry, blood pressure, body
composition and fitness.
Discussion: Water-based versus land-based exercise elicit different haemodynamic responses and, consequently, distinct patterns of shear stress in arteries, including
those supplying the brain. If the exercise programs prove beneficial, this will inform future strategies for the prevention of cerebrovascular decline in the ageing
‘healthy’ population.

1. Introduction
The impact of dementia in Australia is considerable, with approxi-
mately a quarter of a million sufferers and 1300 new cases diagnosed
per week (Access Economics, 2009). Dementia is now the second
leading cause of death in Australia and, based on current trends, de-
mentia may overtake heart disease as the leading cause of death in
Australia by 2021 (Australian Bureau of Statistics, 2015). By 2050,
prevalence is expected to reach one million sufferers and 7400 new
cases per week (Access Economics, 2009). Healthcare expenditure to
combat dementia has been projected to surpass that for all other health
conditions by 2060 (Access Economics, 2009).
Pharmacological approaches to the prevention of dementia and
cognitive decline have, to date, been relatively ineffective (Ihl et al.,
2015). Somewhat in contrast, regular exercise reduces cerebrovascular
events (Vogel et al., 2009) and can improve cognition and mental
health in older adults, including those with cognitive impairment
(Hillman, Erickson, & Kramer, 2008; Lautenschlager, Cox, & Kurz,
2010). In older adults at risk of Alzheimer's Disease, the significant
effects of physical activity on cognition from a 24 week home-based
walking program exceeded those previously reported in clinical trials
with cholinesterase inhibitors (Lautenschlager et al., 2008). This study
showed a persistent benefit on cognition twelve months after the active
intervention was completed. Despite these promising findings regarding
the effects of exercise, the mechanisms underlying the cerebral benefits
of physical activity remain largely unknown. Consequently, there is no

∗
Corresponding author. The University of Western Australia M408, Nedlands, WA, 6009, Australia.
E-mail address: danny.green@uwa.edu.au (D.J. Green).

https://doi.org/10.1016/j.mhpa.2018.10.005
Received 20 August 2018; Received in revised form 15 October 2018; Accepted 19 October 2018
Available online 24 October 2018
1755-2966/ © 2018 Elsevier Ltd. All rights reserved.
D.J. Green et al.
evidence-base for the optimisation of exercise programs which might
delay cerebrovascular aging and cognitive decline in humans. Because
exercise can potentially provide a universal, cheap, and relatively side-
effect-free intervention, this is a critical gap in knowledge.
We propose that exercise exerts some of its beneficial effects on
cognition and cerebrovascular events as a result of improvement in
vascular function. Our previous studies involving the assessment of
peripheral conduit arteries (e.g. brachial, femoral) and micro vessels
have shown enhanced endothelial function and, in particular nitric
oxide bioavailability, as a result of the direct haemodynamic effects of
exercise on the vascular endothelium (Green, Hopman, Padilla,
Laughlin, & Thijssen, 2017; Green, Maiorana, & Cable, 2008; Green,
Walsh, et al., 2004; Maiorana, O'Driscoll, Taylor, & Green, 2003). The
benefits of exercise on the vascular system extend to arteries distant to
the working muscle (Green, Maiorana, O'Driscoll, & Taylor, 2004;
Maiorana et al., 2001; Maiorana et al., 2000; Maiorana et al., 2003;
Walsh, Bilsborough, et al., 2003; Walsh, Yong, et al., 2003). We suggest
that cerebrovascular endothelial function may play a key regulatory
role in neuro/angiogenic coupling, the proliferation and survival of
neurons associated with growth of new blood vessels. In addition, we
propose that exercise exerts some of its beneficial effects on cognition
and cerebrovascular effects as a result of repeated increases in cerebral
blood flow, shear stress and, consequently, NO and vascular endothelial
growth factor (VEGF) production. Interventions which enhance en-
dothelial function should therefore decrease cerebrovascular risk and
dementia in humans.
The proposal that cerebrovascular endothelial function may be
mechanistically implicated in dementia has recently gained support in
animal studies (Chu & Heistad, 2010; Gertz et al., 2006). For example,
Gertz et al., 2006 reported that exercise trained mice exhibit higher
cerebral blood flow in the ischaemic region following stroke than un-
trained animals, along with better cognitive outcomes. These benefits
were not evident in mice treated with an inhibitor of endothelial NO
during exercise. With exercise training, peripheral arteries experience
repeated increases in blood flow and shear stress; the dragging force of
blood across the inner arterial wall. These stresses provide a stimulus to
enhanced endothelial function (Black, Green, & Cable, 2008; Green,
2009; Green et al., 2010, 2017; Green, Spence, Halliwill, Cable, &
Thijssen, 2011; Naylor et al., 2011; Thijssen et al., 2010; Tinken et al.,
2010). If this is also true in the cerebral circulation, then repeated
episodic increases in brain blood flow that accompany exercise should
improve cerebrovascular function and promote healthy cognitive aging
(Green, Maiorana, et al., 2004; Maiorana et al., 2001; Maiorana et al.,
2000; Maiorana et al., 2003; Walsh, Bilsborough, et al., 2003; Walsh,
Yong, et al., 2003). During acute bouts of exercise, cerebral blood flow
velocities increase (Ogoh & Ainslie, 2009a, 2009b). In addition, resting
cerebral blood flow is elevated by habitual exercise across the lifespan
(Ainslie et al., 2008) and a small number of studies have suggested that
exercise training may modify cerebral vascular function (Akazawa
et al., 2012; Murrell et al., 2013). Although these data suggest that
exercise is associated with enhanced cerebrovascular blood flow,
longitudinal studies, and in particular randomised controlled trials
pertaining to the impact of exercise training on cerebrovascular func-
tion in humans, are sparse.
One form of exercise that may provide a novel, yet highly achiev-
able, stimulus for cerebrovascular benefit is water immersion-based
exercise. Due to hydrostatic effects, water immersion in the upright
posture is associated with a cephaloid shift in blood volume, increased
venous return, preload and stroke volume (Hall, Bisson, & O'Hare,
1990; Weston, O'Hare, Evans, & Corrall, 1987). Increases in cardiac
output of ∼50% have been reported (Weston et al., 1987). Immersion
of the lower body in water increases cerebral blood flow, both at rest
and during exercise (Carter et al., 2014; Pugh et al., 2015). We recently
completed a study in which healthy participants were submerged to the
level of the right atrium in water whilst continuous measurements of
brain blood flows were collected (Carter et al., 2014). Water immersion
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Mental Health and Physical Activity 15 (2018) 153–163
increased transcranial Doppler measures of brain blood flow velocity in
cerebral arteries, which was reversible when water was drained from
the immersion tank. Water immersion therefore increases brain blood
flow and we hypothesise that repeated immersion may lead to adap-
tations which enhance cerebrovascular function in humans. It is also
germane that water based exercise is seen as highly feasible in older
populations, due to the weight assistance and the minimisation of falls
risks. We propose to utilise this novel effect of water immersion to
manipulate (i.e. increase) cerebral shear stress and blood flow during
each bout of exercise, thereby optimising impacts on cerebrovascular
health whilst simultaneously decreasing musculoskeletal impact and
risk of injury.
The Preventia Study is a randomised controlled superiority trial of
the impact, in older adults, of a novel aquatic exercise intervention
which aims to optimise the haemodynamic stimulus responsible for
improvement in cerebrovascular function in humans. The study em-
ploys techniques that we have developed and validated which reveal
changes in brain blood flow and its regulation in humans. This inter-
vention will establish the impact of exercise training on cerebrovascular
function and its association with cognition in older individuals with
memory concerns. This project is clinically relevant, with potential to
address the future impact of a common, debilitating and costly disease,
and with potential carryover benefits to other conditions such as car-
diovascular disease.
2. Methods/design
2.1. Hypotheses
1. A land-based walking program will improve cerebrovascular func-
tion in older participants (≥50 years), relative to a matched but
non-trained control group.
2. Water-based walking, involving elevated responses to the circula-
tion (e.g. cerebral blood flow and shear stress) when training in
water, will improve cerebrovascular function to a greater degree
than land-walking.
3. Improvement in cerebrovascular measures will be significantly re-
lated to changes in cognition, memory and clinical measures.
2.2. Participant recruitment
The study was approved by the Human Research Ethics Committee
of The University of Western Australia and is a registered clinical trial.
All participants provided written, informed consent prior to participa-
tion. This study conforms to the standards set by the Declaration of
Helsinki.
Cognitively healthy older participants were recruited. Participants
were subjective memory complainers (in the absence of depression or
dementia), one of the clinical groups considered at increased risk of
future cognitive decline. Recruitment was undertaken through a com-
bination of approaches including radio appeals, newspaper advertise-
ments, approaches to aged groups, publications and community cen-
tres. Six overlapping cohorts were recruited for the study, with baseline
testing commencing in January 2014 and finishing in May 2017. The
study involved baseline screening and testing, followed by a 24 week
intervention (land-based or water-based walking, or control group),
repeat testing, a further 24 weeks of no contact with the university,
then a final test battery at 48 weeks (Fig. 1.)
2.2.1. Inclusions
To be eligible, participants were 50 years or older (females were post-
menopausal); healthy but inactive (less than 60 min of moderate or higher
intensity exercise per week, for at least three months), with subjective
memory complaints but no diagnosed cognitive impairment; available for a
15 month period; and prepared to undertake 150 min per week of mod-
erate intensity exercise for six months, in a centre-based program.
D.J. Green et al. Mental Health and Physical Activity 15 (2018) 153–163

Fig. 1. Preventia study structure and timetable.

2.2.2. Exclusions
Potential participants were excluded if they had a diagnosed cog-
nitive impairment (mild cognitive impairment or dementia), or if they
fell into the following categories based on baseline screening tests:
Modified Telephone Interview Cognitive Status – TICS-m (Welsh,
Breitner, & Magruder-Habib, 1993) less than 32, Geriatric Depression
Scale - GDS (Yesavage et al., 1982) greater than six, Standardised Mini-
Mental State Examination - sMMSE (Folstein, Folstein, & McHugh,
1975; Molloy & Standish, 1997) less than 24, Repeatable Battery for the
Assessment of Neuropsychological Status (RBANS) delayed memory
index score more than 1.5 standard deviations below the age-related
mean. Other exclusion criteria comprised: letter not returned by the

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relevant General Practitioner (GP) to confirm no objection to partici-
pation; participant had smoked tobacco in the last 12 months; systolic
blood pressure greater than 160 mmHg or diastolic blood pressure
greater than 100 mmHg; weekly alcohol intake greater than 280 g per
week and/or drinking more than 40 g ethanol in one session; body mass
index (BMI) greater than 40 kg/m2; total cholesterol greater than
7.0 mmol/L; taking psychotropic medications such as cognitive en-
hancers, anti-epileptics or lithium; intermittent use of non-steroidal
anti-inflammatory drugs; not able to meet the requirement of moderate
physical activity; not confident of exercising in chest-deep water; pos-
sible hospital admission or period of treatment/rehabilitation in the
next six months; simultaneous participation in another research trial;
lack of fluency in English; and past or present history of medical con-
ditions that were likely to preclude moderate intensity exercise or
which were likely to compromise survival (for a full list, see the trial
registration details).
Upon contact from a potential participant, an initial, brief phone
interview was conducted in order to explain the study more fully, and
to screen out clearly ineligible participants or those who were not in-
terested in participating further (Fig. 2), for example those who were
too active or had an established medical condition that could interfere
with their ability to participate in the study testing or in the exercise
sessions.
Of 911 initial contacts (Fig. 2), 241 progressed to a second and more
extensive and formalised phone screening interview (45–60 min dura-
tion), which included questions related to memory, current physical
activity, general health, alcohol, medications, and medical history, as
well as questionnaires in relation to cognition and mood; the TICS-m
and GDS. This process led to further participants being deemed in-
eligible, as per the reasons provided in Fig. 2. During the phone screen,
subjects were asked specific questions regarding their memory, (“Do
you have difficulty with your memory”, “Do you think your memory is worse
than it should be for your age”, “Have you ever had an assessment of your
memory”). Negative responses to the first question represented an ex-
clusion criterion. There was no specific time period mentioned. More
formally, the MAC-Q assessed self-perceived memory decline.
2.3. Screening visit
Subsequent to a successful phone interview, 114 eligible and willing
participants progressed to an initial, fasted, screening visit, of ap-
proximately 2 h duration (Fig. 1). The study was explained in detail,
and a written informed consent was obtained for participation in the
study. Consent to contact the participant's GP was also obtained. Each
participant was provided with a letter for their GP which explained the
study protocol, and requested communication of any concerns re-
garding the patient's participation, and also release of any relevant
medical history details. Approval for participation from the GP was
required for enrolment into the study.
Following the consent process, height, weight and anthropometric
measures were taken using standard techniques, after which the par-
ticipant underwent a resting (supine) 12-lead ECG (XScribe, Mortara
Instrument, Milwaukee WI, USA). This was followed by blood pressure
recording, undertaken supine in a quiet darkened and isolated room,
with a cuff auto-inflating every 2 min for 20 min (Dinamap V100, GE
Healthcare, USA). Blood was sampled, after which the participant was
given a light breakfast (muesli or toast; tea or coffee). Cognitive testing
was then conducted in a separate room, with a trained psychologist or
psychology researcher conducting the tests under supervision; a self-
reported, detailed demographic and lifestyle questionnaire was then
also completed (details below). The test battery took approximately
90 min to complete. The psychology researcher was blinded to the in-
tervention group allocation. Subsequent to the screening appointment,
the ECG recording and biochemistry results were evaluated by a med-
ical practitioner affiliated with the study. Any results of concern were
communicated to the study coordinator and then to the participant's
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Mental Health and Physical Activity 15 (2018) 153–163
GP, and the participant was excluded or further information sought
before enrolment, depending upon the particular condition/result de-
tected.
2.4. Further baseline testing sessions
If the participant remained eligible after the screening visit (Fig. 1),
four further testing sessions were conducted prior to formal enrolment
in the study. Specific details are given below, but briefly; the famil-
iarisation visit included a second, 20 min blood pressure assessment as
described above; familiarisation with the mouthpiece and treadmill as
per the later fitness test assessment; and familiarisation with the lower
body negative pressure chamber (LBNP), which was to be used in later
cerebral blood flow testing. In addition, a saliva sample was taken to
permit analysis of the ApoE genotype if the participant consented (lack
of consent did not exclude participation in the study). A home blood
pressure monitor was provided to the participant, along with detailed
instructions and a supervised practice. An accelerometer, used to record
general physical activity levels, was also provided and its use explained
at this visit.
The remaining three visits included a DXA (dual X-ray absorptio-
metry) scan for body composition assessment, 12-lead ECG stress test
supervised by a qualified medical practitioner, which also permitted
analysis of aerobic fitness via maximal oxygen consumption (VO2), and
sessions for analysis of limb and cerebral blood flow under a range of
conditions, using advanced imaging techniques, as described in detail
below. Adverse findings or difficulty tolerating the tests during any of
these visits could result in cessation of participation in the study, with
medically relevant data communicated to the participant's GP by the
study coordinator.
2.5. Randomisation
Once all of the baseline testing had been completed, 72 participants
(19 male, 53 female) remained eligible and interested in participating.
These participants were randomised to one of three study groups – land-
based walking, water-based walking, and control (education only)
group (Fig. 2). Stratified block randomisation was used to minimise the
effects of gender. Other known risk factors such as age will be con-
trolled in the analysis. The block randomisation was done using a da-
tabase based on the ‘ralloc’ package within strata. The randomisation
used three randomised block sizes and simple randomisation within
blocks to ensure a ratio of study: control participants close to unity
within strata and overall. The randomisation used a 2 × 3 design so
there were six strata (from one stratification variable and three blocks).
The order of the block size was randomised.
Group assignment was concealed in opaque envelopes and drawn by
a person independent from the study. To maintain concealment of the
allocation the list was kept by one Chief Investigator not blinded to the
group allocation.
Group sizes were Control: 23 (6 male); Land: 24 (6 male) and Water
25 (7 male.) Baseline characteristics for each group are presented in
Table 1.
The control group was asked to maintain their usual levels of ac-
tivity (all participants were inactive at recruitment), and attended the
university every six weeks to participate in an educational seminar of
approximately 1 h, on a topic which was expected to be of interest but
was unrelated to physical activity, for example, “Staying Healthy”,
“Ergonomics at work and home”, “Basic First Aid and CPR”, “Keeping
accident free, Sleep and Health”. The control group attended these
sessions to maintain contact with the research team and to minimise the
possibility of a Hawthorne effect. The two exercise groups (land- and
water-based walking) completed three exercise sessions per week for 24
weeks, building to a 1-h duration over the course of the study (see
below).
D.J. Green et al. Mental Health and Physical Activity 15 (2018) 153–163

Fig. 2. Participant recruitment, inclusion/exclusion, and intervention numbers.

2.6. Exercise training protocols
All exercise was supervised and centre-based, with intensity mon-
itored using a heart rate watch (Polar RS300X HR monitor, Polar
Electro Oy, Finland) and with sessions conducted in small groups in the
early morning and evening. A supervised warm-up and cool down were
included in each session. Land-based walking took place in the uni-
versity grounds as well as along the adjoining river foreshore and
parkland, or on a treadmill if weather indicated. Water-based walking
was in a heated (30 °C) chest deep swimming pool (20 × 30 m) located
at the university. No swimming was involved; water walking was at a
pace that kept the heart rate in the required and monitored target zone.

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D.J. Green et al. Mental Health and Physical Activity 15 (2018) 153–163

Table 1 24 week testing.

Baseline characteristics of the Control, Land-based and water-based walking
groups. 3. Outcome measures in detail
Control Land-walking Water-walking
In order to standardise the time of day of testing across the three
n = 23 (6M, 17F) n = 24 (6M, 18F) n = 25 (7M, 18F) major recall time points (baseline, 24 and 48 weeks), as well as the
interim 12 week blood flow testing session, all test sessions (apart from
Mean (SD) Mean (SD) Mean (SD)
the fitness test and cognitive tests) were conducted in the
Age (yrs) M 64.9 (9.3) 61.3 (6.6) 61.6 (5.6) Cardiovascular Research Laboratory at the University between 6am and
F 61.1 (6.0) 63.3 (7.2) 62.9 (7.2) 11am. Cognitive testing was in the same time slot, but in a quiet room
Ht (cm) M 180.0 (6.1) 173.9 (5.0) 175.4 (4.2)
in a building adjacent to the laboratory. The fitness test was conducted
F 163.4 (6.2) 162.0 (6.5) 163.6 (5.1)
Wt (kg) M 88.5 (15.2) 84.0 (4.2) 94.0 (16.6) in the afternoon, generally between 1pm and 6pm, and used the same
F 68.5 (8.4) 71.0 (10.9) 70.1 (17.0) laboratory facility and the same treadmill throughout the course of the
BMI (kg/m2) M 27.6 (6.1) 27.8 (1.2) 30.5 (5.0) study.
F 25.7 (3.2) 27.1 (3.9) 26.0 (5.4)

3.1. Biochemistry

Duration and intensity of the exercise sessions increased over the
course of the study – from an initial 15 min of exercise, increasing to
50 min by the end of the 24 weeks. Participants (land and water) were
given a personalised target heart rate range, based on heart rate reserve
(HRR) calculated from their initial peak exercise test results. HRR takes
into account differences between subjects in resting heart rate, and has
been recommended in preference to %HRmax by the American College
of Sports Medicine (Garber et al., 2011).
The relationship between heart rate and oxygen consumption
during exercise is maintained for water-versus land-based exercise
(Pugh et al., 2015). Heart rate reserve was therefore used to standardise
exercise intensity for the land- and water-groups. Each exercise session
was guided by an accredited exercise scientist/physiologist (accredited
by Exercise and Sports Science Australia), and heart rate was monitored
and recorded every 5 min throughout the session.
The second session of each week was an interval training session,
with the other two sessions of continuous intensity. The intensity of
training started at 40–45% heart rate reserve in accordance with re-
commendations of the American College of Sports Medicine (Pescatello
& American College of Sports Medicine, 2014), building to 55–65% by
week 24. Additional, ‘make-up’ sessions were provided if required, to
ensure optimal adherence to the protocol.
Mean training intensity expressed as percent heart rate reserve was
calculated from the heart rates taken during the main set of the training
session (i.e. all heart rates except those taken during the warm up and
cool down) and resting heart rate taken from the baseline blood pres-
sure assessment. Adherence to the training program was calculated
from the number of sessions completed out of the number prescribed
(72) expressed as a percentage.
2.7. Repeat laboratory testing
After the initial 12 weeks of exercise/seminars, participants re-
turned for limb and cerebral blood flow analysis tests only. These were
conducted on non-exercise days. After 24 weeks, most of the initial tests
were repeated, as per Fig. 1. Exceptions were the tests which had been
included for screening purposes only (e.g. resting ECG) or those that did
not require repetition (DNA.)
Following the 24 week intervention, the control group was asked to
maintain their current level of physical activity for a further 24 weeks,
but there was no attendance at the university during that time nor was
there deliberate or regular contact between the study staff and parti-
cipants. The two groups who had undergone exercise interventions
were free to continue to exercise or not (they were not specifically in-
structed to continue, nor to desist), at whatever level they chose. This
was a deliberate strategy as it was of interest to ascertain whether the
established patterns of physical activity between weeks 0 and 24 would
be voluntarily maintained.
A final round of testing was conducted at 48 weeks, identical to the
Blood was drawn from the antecubital fossa using a 21G needle by a
trained phlebotomist in the university laboratory into 6 collection tubes
(all Vacuette by Greiner bio-one, Kremsmünster, AT). Sterile techniques
were applied, identical to those used in routine clinical services, and a
maximum of 20 mL of blood was drawn per visit.
Three blood tubes (lithium heparin, fluoride oxalate and EDTA)
were analysed by a commercial pathology laboratory, for lipids (total
cholesterol, triglycerides, low- and high-density lipoprotein (LDL and
HDL), and the ratio of total cholesterol:HDL)), urea and electrolytes
(sodium, potassium, bicarbonate, urea, creatinine, and estimated glo-
merular filtration rate (eGFR)), blood glucose, haematology (hae-
moglobin, red cell count, haematocrit, mean corpuscular volume, mean
corpuscular haemoglobin, mean corpuscular haemoglobin concentra-
tion, red cell distribution width, platelet count and mean platelet vo-
lume, white cell count and differential cell count - neutrophils, lym-
phocytes, monocytes, eosinophils, basophils).
Serum (serum separator tube) and plasma (EDTA and lithium he-
parin) samples were collected for storage at −80 °C in 1 mL aliquots.
All tubes were left at room temp until centrifugation and serum tubes
were left to clot for at least 20 min, and all four tubes were centrifuged
together at 21 °C, for 10 min at 3500 RPM. Platelet parameters (sodium
citrate tube) were analysed in house as described elsewhere (Haynes
et al., 2018).
3.2. Anthropometry
Height was recorded using a wall-mounted stadiometer, with the
head in the Frankfort plane and the participant barefooted. Digital
scales (CPWplus-200, Adam Equipment, Oxford CT, USA) were used to
measure body mass, with the same scales used throughout the course of
the study. BMI was calculated from these values, with the initial
(baseline) height measurement carried forward for BMI calculations
throughout the course of the study. Arm (non-dominant side), waist and
hipgirths were measured with a constant tension measuring tape
(Lufkin W606PM, Cooper Industries, USA); the sequence of measure-
ments was conducted three times and the waist-hip ratio was calculated
for each sequence. The median value was used as the outcome measure.
3.3. Body composition
Body composition was measured using dual X-ray absorptiometry
(DXA - Lunar Prodigy Advance, GE Healthcare, Madison, WI, USA.).
Radiation exposure was 0.8 μSv per scan (2.4 μSv over the study
period.) This is equivalent to about 1/1000th of the background ra-
diation from living in Perth (the city in Western Australia which the
study was conducted) for one year. Standard calibration and quality
assurance procedures were used, in line with the equipment doc-
umentation (www3.gehealthcare.com). Outcome measures were re-
gional and whole body percent fat; grams of fat tissue, lean tissue and

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D.J. Green et al.
bone mineral content; fat mass ratios (trunk:total, legs:total;
(arms + legs)/trunk) and bone mineral density.
3.4. Blood pressure – laboratory
Blood pressure was recorded using an automated sphygmoman-
ometer (Dinamap V100, GE Healthcare, USA) with readings taken in
the supine position, every 2 min for 20 min. Appropriate cuff sizes were
used, based on arm girth measurement, and the non-dominant arm was
cuffed. The test was administered in a quiet, temperature-controlled
room, with the investigator close-by but outside the closed door, and
the display was angled away from the participant's view.
3.5. Blood pressure – home recording
Home blood pressure was recorded on three occasions per time
point, on alternate days (two weekdays and one weekend day) between
4pm and 6pm, prior to the evening meal. No caffeine (tea, coffee,
chocolate) or alcohol was consumed in the 2 h interval prior to the test,
and there was at least 24 h between any moderate or higher intensity
exercise session and the blood pressure measurement. The participant
was instructed to be in a quiet area, not reading, talking, listening to
media or watching television. Measurement was taken while seated,
with the arm supported (bench or pillows) at approximately heart level.
The cuff was placed on the non-dominant arm, and remained in place
during a period of 5 min rest plus five readings commencing at 2 min
intervals. Where possible, the identical monitor was provided for the
participant at each time point. Systolic and diastolic blood pressure
were recorded, together with heart rate. The monitors used (UA-767PC
automated home blood pressure monitor - A&D Medical, Thebarton,
Australia) have been shown to have good validity and reliability
(Rogoza, Pavlova, & Sergeeva, 2000).
3.6. ApoE4 genotyping
A saliva sample was collected according to manufacturer's instruc-
tions during baseline screening, to enable determination of ApoE4
genotype (Genotek, Oragene DNA Self-Collection kit OG-500 - Genotek
Inc, Ottawa On Canada, K2K 1L1). Participants refrained from eating,
drinking, smoking or gum chewing for 30 min prior to the test.
Unwillingness to provide a DNA sample did not exclude participation in
the study. The samples were batched for analysis. They were stored in a
secure location at room temperature (range 15–30 °C).
3.7. Resting ECG
A 12-lead, resting ECG (X-Scribe, Mortara Instrument, Milwaukee
WI, USA) was performed at the initial screening visit only. The parti-
cipant was in a supine posture in a quiet room. The resulting trace was
assessed by a medical practitioner affiliated with the study, and any
issues of concern were referred to the participant's GP, with the parti-
cipant generally excluded, depending upon the severity of the ECG
abnormality.
3.8. Graded exercise stress test
Aerobic fitness was assessed via using a graded exercise test on a
treadmill. In line with ACSM recommendations at the time of the study
launch (Gordon & Pescatello, 2009), baseline and 12 month tests were
medically supervised, and included 12-lead ECG monitoring. Medical
supervision was not required at the six month test, because of the recent
initial supervised test. However, the 12 month repeat test was medically
supervised, again in keeping with guidelines (Pescatello & American
College of Sports Medicine, 2014).
At baseline this exercise stress test served the dual purpose of fitness
test and screening for cardiovascular risk assessment. Results of the test
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Mental Health and Physical Activity 15 (2018) 153–163
were assessed by a cardiologist affiliated with the study, and clearance
was required for enrolment in to the study proper. If any medical or
ECG issues of concern were apparent, the participant's GP was notified
and the results supplied to facilitate any follow-up.
Prior to the exercise test the participant had abstained from food
and drinks other than water, for 2 h. Standard preparatory techniques
were used for the ECG, with light abrasion of the skin to ensure good
contact. An initial resting (supine) trace was recorded to confirm a
normal ECG with no artefact. A blood pressure cuff requiring manual
inflation was fitted to the left arm, and the participant was asked to
stand on the treadmill. Resting (upright) heart rate and blood pressure
were recorded, and the Borg scale for rating of perceived exertion
(Borg, 1982) was explained. The participant applied a nose clip and
inserted a Hans Rudolph mouthpiece, as per the familiarisation session.
The mouthpiece had a two-way valve which permitted subsequent
analysis of expired air. One minute of respiratory data was collected
with the participant standing on the stationary treadmill, to ensure
values gained were valid and accurate. The treadmill starting speed and
gradient were set (Table 2) and the ECG recording was started; once the
participant was ready the test commenced. The protocol comprised
continuous, incremental stages lasting 3 min per stage - see Table 2 for
speed and gradient specifications.
During the medically supervised tests, the ECG and heart rate were
continuously monitored by the supervising GP and by an exercise
physiologist. Blood pressure was measured manually by the supervising
GP in the final minute of each stage. Heart rate and RPE were recorded
in the last 30s of each stage.
Verbal encouragement was provided throughout the test, to elicit
maximal effort. The test ended once the participant was no longer able
to achieve the required workload, i.e. volitional exhaustion was
reached. Participants were able to terminate the test at any time by
pressing the emergency stop button or by signalling by hand that they
wanted the test to stop. If any contraindications to the test were ob-
served by any of the researchers, they were under instruction to stop the
test immediately (e.g. ECG concerns, an exaggerated hypertensive re-
sponse, chest pain or angina), in line with ACSM guidelines.
On completion of the test, blood pressure and heart rate were
monitored for a minimum of a further 6 min, with the participant su-
pine, to ensure that ECG, blood pressure and heart rate responses in
recovery were normal, returned to baseline levels, and the participant
was comfortable and asymptomatic.
3.9. Physical activity
Representative weekly physical activity volume was collected using
an Actigraph accelerometer (Actigraph GT1M, Pensacola, FL, USA),
attached to an elasticised strap. The device was placed over clothing, on
the right hip bone. Information was collected at entry, 24 and 48 weeks,
to determine any changes in lifestyle physical activity which occurred
around the prescribed centre-based activities. A continuous eight-day
diary was used to record hours of wear.
Table 2
Protocol for aerobic fitness test.
Stage Speed (km/h) Gradient (%)
1 2.4 3
2 3.2 4
3 4.0 5
4 4.8 6
5 5.6 8
6 6.4 10
7 8.0 10
8 9.6 10
9 11.2 10
10 11.2 15
D.J. Green et al.
3.10. Demographic, health, lifestyle and medication information
This data was obtained via questionnaire, in the same session as the
cognitive assessment. Questions encompassed living arrangements,
years of education, occupation, income, computer use, country of birth,
clubs, hobbies, family medical history, and minimal diet/nutrition in-
formation (serves per day, supplement use), as well as alcohol con-
sumption, smoking history, medical history, medications, physical ac-
tivity and exercise history.
3.11. Cognition
A battery of relatively brief but reliable and well-validated cognitive
tests was administered at baseline, 24 and 48 weeks, to encompass a
range of cognitive domains. These tests were conducted by a trained
clinical or research psychologist, who was blinded to the treatment
allocation group. A second research psychologist independently scored
all test results a second time to ensure accuracy and consistency. All
testing was administered in a quiet room away from the laboratory. In
addition to the TICS-m and GDS conducted during the initial phone
screening procedures, the following tests were conducted: the stan-
dardised Mini Mental State Examination (sMMSE); the Repeatable
Battery for the Assessment of Neuropsychological Status; the Trail
Making Test (TMT) Parts A + B; the Hospital Anxiety and Depression
Scale; the 5-item World Health Organization Well-Being Index (WHO-
5); and the Memory Complaint Questionnaire (MAC-Q). The demo-
graphic, WHO-5, HADS and MAC-Q questionnaires were self-adminis-
tered, whilst the other tests were all researcher-administered.
• sMMSE - The SMMSE is a brief cognitive test with a maximum score
of 30. The test is widely used by physicians and as a screening test
for cognitive impairment. Items on the sMMSE assess word recall,
orientation to time and place, attention, language and visuospatial
abilities (Folstein et al., 1975; Molloy & Standish, 1997).
• RBANS – is a brief (approx. 30 min), reliable battery designed to
assess cognitive decline or improvement across multiple areas of
cognitive function, including immediate and delayed memory, at-
tention, language and visuo-spatial skills. Specific tests capture
episodic memory (list learning and story recall), speed of informa-
tion processing (digit symbol coding), language abilities (picture
naming and semantic fluency), visuo-construction skills (figure copy
and line orientation) and executive functions (fluency, digit span).
The RBANS is suitable for use in adults < 89y, is sensitive to a wide
range of cognitive abilities, and is increasingly used in clinical trials
of treatments that impact neurocognitive status (Randolph, Tierney,
Mohr, & Chase, 1998).
• TMT Parts A + B – indexes individual abilities related to visual
search, scanning, speed of processing, mental flexibility, and ex-
ecutive functioning. It contains two parts: TMT-A requires partici-
pants to draw lines in the correct sequence connecting 25 encircled
numbers, distributed on a sheet of paper. The TMT-B is similar,
except participants are required to alternate between numbers and
letters. Scoring is based on the amount of time required to complete
each task (Army Individual Test Battery, 1944).
• HADS – this test is a widely used measure of psychological distress,
designed for use in non-psychiatric patient populations (Zigmond &
Snaith, 1983). The HADS consists of two 7-item subscales, mea-
suring anxiety and depression respectively; though latent structure
analysis suggests it is best used as a more general measure of dis-
tress. (Cosco, Doyle, Ward, & McGee, 2012). The HADS was in-
cluded as a potential covariate, given that individual difference in
distress may influence cognitive performance.
• WHO-5 Well-Being Index – is among the most widely used measures
to assess subjective psychological well-being. It uses a generic global
rating scale, which only contains positively phrased items.
Participants are asked to rate how well each of the 5 statements
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Mental Health and Physical Activity 15 (2018) 153–163
applies to him/her when considering the last 14 days, on a scale
from 5 (all of the time) to 0 (none of the time). Thus, the raw score
ranges from 0 (absence of well-being) to 25 (maximal well-being).
(Staehr, 1998).
• MAC-Q Memory Complaint Questionnaire provides a brief assess-
ment of subjective memory complaints. It consists of five items as-
sessing memory in specific situations and one item measuring
overall self-perceived memory decline. Drawing on earlier studies, a
score of 25 and above on the MAC-Q is taken to be indicative of
significant memory complaint, potentially requiring further assess-
ment.(Crook, Feher, & Larrabee, 1992).
3.12. Cerebrovascular function
This was assessed by combining bilateral measures of cerebral ar-
tery blood flow velocity using non-invasive, transcranial Doppler
techniques according to standardised approaches (Willie et al., 2011).
In addition to resting baseline intra-cranial velocity recordings, three
test conditions were implemented to challenge the cerebrovascular
system and provide information as to the functional responsiveness to
distinct types of stimulation. These test conditions reflected neurovas-
cular coupling, dynamic cerebral autoregulation, and cerebrovascular
CO2 reactivity (described in more detail below). Intra-cranial velocity,
as an index of cerebral blood flow, was assessed at baseline, 24 and 48
weeks, with an additional assessment half way through the exercise
intervention period (at 12 weeks.) These tests were conducted on non-
exercise days, at the same time of day, and the participants were re-
quired to have abstained from moderate/vigorous physical activity,
caffeine and food, for at least 6 h prior to the test. Previously published
guidelines were followed (Willie et al., 2011) so the procedures will be
described in brief here.
At the start of the session, the participant was fitted with a head
frame (Marc 600, Spencer Technologies, Seattle WA, USA) similar to a
bicycle helmet frame, with a 2-MHz ultrasound probe located on each
temple, to facilitate imaging of the cerebral arteries. A Finometer PRO
(Finometer, Finapres Medical Systems, Amsterdam, The Netherlands)
allowed continuous monitoring of beat-to-beat blood pressure via
photoplethysmography. Following instrumentation, the participant lay
supine for the remainder of the test period, with the lower half of the
body enclosed in a lower body negative pressure chamber (LBNP).
Although this chamber was only activated for one test condition,
standardising the participant's position throughout the entire test pro-
tocol ensured optimal reproducibility of blood vessel images. Once the
participant was in position, a facemask which spanned the mouth and
nose was fitted and this mask was used during baseline recording and
all three test conditions. A 10 min stabilisation rest period commenced,
after which 10 min of continuous recording was conducted to provide
baseline blood flow velocity measures (LabChart 7 software, AD
Instruments, Sydney, Australia.) The following tests were then con-
ducted, always in the same order. There was a rest period of at least
10 min observed between each of these tests. On the left side, the
middle cerebral artery (MCA) was insonated, whilst on the right side
the posterior cerebral artery (PCA) was assessed. This remained the
same for all tests and for all subject attendances. The same sonographer
completed all of the following tests in a given subject.
• Neurovascular coupling - neural activation of the brain triggers a
cerebral blood flow response. In the ageing brain and various
pathologies (Phillips, Chan, Zheng, Krassioukov, & Ainslie, 2016),
some ‘uncoupling’ of this response can occur (Venkat, Chopp, &
Chen, 2016). Cerebral blood flow was assessed in response to in-
creased neural activity induced by repeated trials of voluntary eye
movement (tracking) and cognitive tasks (a reading exercise). Re-
sponse times to these tests and the magnitude of change in brain
blood flow provide indices of cerebral neurovascular function. For
this test, the participant breathed room air through the facemask,
D.J. Green et al.
while the research assistant held reading material in a comfortable
position for the participant. The participant spent 2 min with their
eyes closed, then 2 min with their eyes open (reading). This was
immediately followed by five sets of 30s with eyes closed followed
by 30s of reading, for a 9 min total protocol. This methodological
approach is in accordance with published guidelines on this tech-
nique (Phillips et al., 2016).
• Dynamic cerebral autoregulation – for this test, cerebral blood flow
was assessed in response to different ‘doses’ of blood pressure ma-
nipulation. Alterations in blood pressure were induced by an oscil-
lating vacuum pressure around the lower half of the body (to the
level of the umbilicus), via a LBNP chamber (Vacustyler –
Weyergans, Dueren, Germany). Impairment in dynamic cerebral
autoregulation is strongly linked to adverse clinical outcomes (Aries,
Elting, De Keyser, Kremer, & Vroomen, 2010; Tzeng & Ainslie,
2014). After 5 min of baseline data collection, the LBNP was acti-
vated in a 5s-on/5s-off protocol with a suction pressure of 90 mbars.
This protocol was continued for 2 min (12 cycles.) After 2 min rest,
the interval was switched to 10 s on/off, and data for 12 further
cycles (4 min) were recorded. This methodological approach is in
accordance with published guidelines on this technique (Meel-van
den Abeelen et al., 2014). We opted to use LBNP to assess auto-
regulation because it involves supine assessment which may be
more appropriate and manageable for repeated assessments in our
older patient population. We also considered that the pressures
could be more accurately standardised for repeated assessment
using LBNP. Finally, we considered that an exercise stimulus (e.g.
the squat-stand), might be affected by metaboreflexes (Braz et al.,
2014), and may therefore not have been an ideal repeated measures
tool for pure autoregulation assessment in this exercise training
study.
• Cerebrovascular CO2 reactivity – this test measures a dose-response
effect to different ‘doses’ of blood CO2, achieved by switching from
room air to gas mixtures that contain differing concentrations of
CO2. Impairment in cerebrovascular CO2 reactivity is an in-
dependent predictor of stroke risk and cognitive impairment
(Markus & Boland, 1992; Markus & Cullinane, 2001). The partici-
pant initially breathed room air for a 5 min baseline collection
period. The mask was then connected to a Douglas bag containing a
mixture of 3% CO2, 21% O2 and balanced N2. The participant con-
tinued to breathe normally for a further 4 min. The mask was then
immediately connected to a second Douglas bag containing a mix-
ture of 6% CO2, 21% O2 and balanced N2. . Four minutes of data
collection were recorded for this concentration, after which the
participant was allowed to rest and the instrumentation was re-
moved. Previously published guidelines were followed (Willie et al.,
2011).
3.13. Limb blood flow and arterial structure
In addition to the structure and function of the cerebral arteries,
those of the conduit arteries (brachial and femoral) in response to the
land and water-based exercise programs were assessed. High-resolution
ultrasound was used to assess arterial wall thickness (brachial, femoral
and common carotid arteries) and FMD of the brachial and femoral
arteries. FMD is a non-invasive measure of endothelial function that is a
marker of cardiovascular disease (Green, Jones, Thijssen, Cable, &
Atkinson, 2011; Rossi, Nuzzo, Origliani, & Modena, 2008). Our as-
sessment and analysis techniques have been published in detail in
previous papers (Thijssen et al., 2011; Woodman et al., 2001).
Prior to the test session, the participant had fasted, abstained from
caffeine and refrained from moderate/vigorous physical activity for at
least 6 h. Blood pressure-lowering- or Viagra-type medication use was
queried, as they are contraindicated for GTN administration. Measures
were assessed at baseline, 12, 24 and 48 weeks, with all tests scheduled
for non-exercise days.
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Mental Health and Physical Activity 15 (2018) 153–163
The participant lay supine in a quiet, temperature-controlled room
for a 20 min rest period. At the end of this period, blood pressure was
assessed using an automated sphygmomanometer (Dinamap, V100, GE
Healthcare, USA) to check for consistency with previous (screening)
blood pressure values for that participant. Baseline, high-resolution
ultrasound scans of the brachial (distal third of the upper arm) and
superficial femoral (upper third of the thigh, near the groin) arteries
were then simultaneously collected by two trained sonographers for
assessment of wall thickness (IMT), using 10-MHz linear array probes
attached to a high-resolution ultrasound machine (T3200, Terason,
Burlington, MA, USA). Camtasia software (Camtasia Studio 8,
TechSmith, Okemos, MI, USA) was used to capture the data for 1 min.
Conduit artery structure and function were measured under three
conditions designed to elicit significant blood flow responses in the
relevant arteries: 1) flow mediated dilation (FMD); the compensatory
arterial diameter response to increased shear stress following a 5 min
period of proximal arterial occlusion induced by cuff inflation; 2)
sublingual administration of a nitric oxide donor and vasodilatory agent
(400 μg glyceryl trinitrate spray – GTN); and 3) response to an ischemic,
handgrip exercise condition.
FMD in peripheral arteries is strongly correlated with coronary ar-
tery function (Takase et al., 1998) and independently predicts cardiac
events in those with cardiovascular disease or risk factors, and also in
asymptomatic individuals (Green, Jones, et al., 2011). A 1% decrease in
FMD is associated with a 13% increase in cardiovascular event risk
(Green, Jones, et al., 2011). FMD was assessed following a 5 min oc-
clusion of blood flow achieved by inflation of the pneumatic cuff
around the forearm and thigh, with the cuff distal to the ultrasound
probe location. A rapid inflation/deflation pneumatic blood pressure
cuff (D.E. Hokanson, Bellevue, WA USA) was placed on the dominant
forearm, immediately distal to the olecranon, but was not inflated. A
second cuff was placed around the thigh approximately 15 cm distal to
the inguinal ligament. Cuffs were simultaneously inflated to 220 mmHg
to restrict arterial flow for 5 min, then rapidly deflated, using an au-
tomatic (Hokanson) cuff inflator. Diameter and flow recordings, via a
high frequency linear array probe, were continuously recorded for
1 min at baseline, during the last 30s of cuff inflation, and for a further
5 min after cuff deflation. Brachial and femoral views were captured
simultaneously by two sonographers highly trained in the imaging of
these arterial parameters.
Following the FMD test, common carotid IMT was assessed.
Thickening of the carotid arterial wall is associated with increased
cerebrovascular (e.g. stroke) and cardiovascular risk (Chambless et al.,
1996; Hollander et al., 2003; Johnsen et al., 2007; Lorenz, Markus,
Bots, Rosvall, & Sitzer, 2007). With the participant supine and the head
slightly extended and rotated, the left common carotid artery was lo-
cated using a 10-MHz linear array probe. Longitudinal images were
obtained from three planes; the optimal angle of incidence and two
complementary angles (anterior, lateral and posterior). The image was
recorded for an interval of 3–5 beats, for a region approximately 1 cm
distal to the bifurcation of the artery. This was repeated for the right
common carotid artery.
Prior to the subsequent (after a 10 min rest period) administration
of the GTN spray, a further blood pressure check was conducted using
the Dinamap sphygmomanometer. One minute baseline scans of bra-
chial and femoral arteries were then recorded simultaneously. A single,
400 μg dose of GTN was then sprayed under the participant's tongue.
Three minutes after administration, simultaneous brachial and femoral
ultrasound recordings were recorded, for a period of 5 min. Blood
pressure was checked again, after which time the participant rested for
15 min, remaining supine, with a further blood pressure check at the
end of this period.
The final limb blood flow assessment comprised an ischaemic ex-
ercise test. This hyperaemic response to ischaemic exercise provides an
index of arterial lumen remodelling (i.e. structural size). The peak hy-
peraemic blood flow response and the peak conduit artery diameter
D.J. Green et al.
response to this stimulus in humans provide valid indices of resistance
and conduit artery size or remodelling, respectively.
For the ischaemic exercise test a cuff was placed distal to the ole-
cranon, and a weighted handgrip exercise device was moved into a
position comfortable for the participant. Following a 1 min baseline
scan, the cuff was rapidly inflated and sustained for 5 min. For the
middle 3 min, the participant performed the handgrip exercise in time
with a metronome (25 contractions per minute) with a load of 0.5 kg.
Upon cessation of the exercise the participant lay as still as possible for
the remainder of the test. Data was recorded for the final 30 s of cuff
inflation, and continuously for the 5 min subsequent to cuff deflation. A
final blood pressure measurement was recorded, and the session ended.
4. Power calculations and planned statistical analysis
The paper of Akazawa et al. (2012) was used as an exemplar for
power assessment. In this study cerebral blood flow velocity (our pri-
mary outcome measure) increased from 62 cm/s before training to
70 cm/s after training, with a standard deviation of 12 cm/s (see Fig. 2
in Azakawa et al. (2012) (n = 10, 60 ± 2 years)). Assuming 80%
power, and α = 0.05 (G*power), these data suggest that a sample size
of 20 subjects is required to achieve statistical significance. Our inter-
vention was of similar intensity as this previous study of exercise, and it
was also of longer duration (24 vs 8 weeks) and centre-based.
The normality of data will be assessed graphically by using histo-
grams and box plots. Continuous variables with normal distribution will
be described using means and standard deviations; median and inter-
quartile range will be used for those without a normal distribution.
Categorical variables will be described using frequency tables.
Outcome measures will be primarily assessed with an intention-to-
treat analysis at the end of the intervention with secondary analysis for
the 12- month time point. This effect will be tested as the interaction
between the allocation group (land-based walking; water-based
walking and control) and time, on the primary and secondary out-
comes. We will apply multilevel regression models (mixed models)
given the repeated measures design. The baseline value of each out-
come will be included in the model as a covariate. Alpha will be set at
0.05 and all statistical tests reported will be two-tailed.
5. Summary
Cognitive decline is a debilitating and costly condition, for which
pharmaceutical interventions are relatively ineffective. In contrast,
exercise may prove an inexpensive, achievable and effective strategy
for preventing or reducing cognitive decline in older adults. Vascular
contributions to cognitive decline including Alzheimer's disease have
been established, and exercise is known to be of benefit for many
vascular structural and functional parameters.
The current study was designed to test two exercise protocols which
will elicit different shear stresses on the arteries of sedentary older
adults with subjective memory complaints. This centre-based, 24 week
program will establish the impact of exercise training on cere-
brovascular function and its association with cognition in older adults.
The project is therefore highly clinically relevant with potential to ad-
dress the future impact of a common, debilitating and costly disease.
Trial registration
ACTRN12614000017628.
Declaration of interest
This research was supported by NHMRC grant 1045204. The
NHMRC had no role in study design, collection, analysis and inter-
pretation of data, writing the report or in the decision to submit the
article for publication.
162
Mental Health and Physical Activity 15 (2018) 153–163
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgements
This work was supported the National Health and Medical Research
Council of Australia (1045204). DG is supported by a NHMRC Principal
Research Fellowship (APP1080914).
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