Experimental design and overview

This randomised trial will follow a two-group parallel design. Participants will be assigned (in a randomised and counter-balanced fashion—see the Randomisation section) to one of two 12-week, work-matched, aerobic exercise training interventions (MICT or HIIT; two groups of n=35). Briefly, the experimental protocol will consist of (1) eligibility screening (t−1), familiarisation of the exercise protocols and baseline testing (t0), (2) a 12-week aerobic exercise intervention with testing at the 6-week mark (t1), (3) post-training testing (t2) and and (4) 12-week follow-up testing (t3) to test the maintenance and short-term robustness of the intervention induced effects (figure 1). The trial will be reported according to the Consolidated Standards of Reporting Trials guidelines. The Standard Protocol Items: Recommendations for Intervention Trials list is detailed in table 1.53 The first participant was recruited in November 2021, and data collection is expected to be completed by late 2023. Study assessments will be conducted over two sites in Melbourne, Australia: Victoria University and the University of Melbourne’s Melbourne Brain Centre Imaging Unit.

Table 1

Standard Protocol Items for Randomised Trials: schedule of enrolment, interventions and assessments

Figure 1

Graphical timeline of the Train Smart Study. HIIT, high-intensity interval training; MICT, moderate-intensity continuous training.

Following screening for eligibility, participants who satisfy the selection criteria will perform three baseline testing sessions. The first session will be a familiarisation session, during which participants will perform an incremental exercise test, and practice sessions of the exercise training intervention and cerebrovascular function tests (~15 min between tests). Additionally, participants will be given a wrist-based activity monitor (GENEActiv, Activinsights) for the valid and reliable assessment of baseline physical activity levels, as previously established.54 Participants will be required to wear the activity monitor for 7 days after the familiarisation session and before the remaining baseline testing sessions. This duration is sufficient to reliably estimate habitual physical activity in adults.55–57 The remaining two baseline testing sessions will be separated by approximately 48 hours and will be performed in the following order: (1) cognitive function tests, mood and health questionnaires and 7T MRI brain scan; (2) resting muscle biopsy, anthropometry assessment, symptom-limited incremental exercise test and the cerebrovascular function tests.

After the initial 6 weeks of training (t1), participants will repeat the 7T MRI brain scan and the mood and health questionnaires, while only completing the CogState component of the cognitive function tests. After the 12-week training intervention is complete (t2), participants will repeat the t0 testing sessions, replicated for order and timing. A final round of testing will be conducted 12 weeks after the end of training (t3), during which participants will repeat the testing sessions from t0 and t2. Physical activity levels will also be measured for a period of 7 days prior to t3 to give an indication of activity levels between t2 and t3. Every attempt will be made to ensure between-participant and within-participant assessments are performed at the same time of day (eg, between 06:00 and 09:00 hours for all blood and muscle analyses, between 09:00 and 17:00 hours for all MRI scans and cognitive function tests) to limit the effects of diurnal variation on these assessments. However, participant and equipment (eg, MRI scanner) availability will vary and ultimately take priority, and all deviations from this schedule will be reported in the final manuscript. Specific details of these protocols are described below.

Participant selection

Seventy sedentary, cognitively normal, middle-aged (45–65 years) adults will be recruited from the general population. A series of prestudy screening questionnaires will be used to ensure that participants meet entry criteria, principally that participants are sedentary and do not meet the Australian Physical Activity Guidelines for Adults (ie, the accumulation of 150 min of moderate-intensity physical activity, or 75 min of vigorous-intensity physical activity, or an equivalent combination of both moderate and vigorous activities, each week) for the previous 6 months. In addition, participants will be enrolled if they have no significant medical conditions precluding participation in an exercise intervention, have no contraindications for MRI, do not have cognitive impairment and do not present with other specified exclusions (table 2). Participants will also be required to obtain a medical clearance from their general practitioner to participate in the study. Cognitive function will be screened by trained assessors using the Montreal Cognitive Assessment, where a score of 26–30 is validated to indicate cognitively normal.58

Table 2

Inclusion and exclusion criteria

Once deemed eligible (as per table 2), participants will provide informed consent by signing a hard copy of a ‘participant information and consent form’. In addition, participants will be asked to sign a hard copy of a ‘consent for future use of data’ form, which provides additional consent provisions for the collection and use of participant data and biological specimens in ancillary studies. JRB and NZ will be responsible for eligibility screening, as well as the collection and secure storage of consent forms.

Randomisation and blinding

Participants will be allocated into two groups by a computerised adaptive randomisation tool developed specifically for this trial using the Pocock Minimisation algorithm (R Studio),59 and developed independently of the research team (ie, sequences are random and concealed from the research team). The randomisation tool will allocate participants via the minimisation of three covariates: cardiorespiratory fitness (above or below age-matched norms),60 age (45–49 years, 50–59 years or 60–65 years) and sex (male, female or other). A researcher unblinded to the participant group (JRB) will be responsible for inputting covariate data into the adaptive randomisation tool prior to allocation, and randomisation will occur after the baseline sessions.

The assessors conducting the cognitive testing, questionnaires and MRI analysis will be blinded to participant group. Participants will be aware of their group allocation based on the exercise-training programme they are administered. To avoid contamination, the exercise sessions for the two groups will be conducted at different times throughout the day, and, if this is unavoidable, then participants from the two groups will be separated within the study site.

Outcomes and analyses

Primary Outcome:

Secondary outcomes:

Between-group difference in global and regional brain blood flow change from t0 to t2.

Between-group difference in brain blood vessel health (eg, intracranial stenoses and pulse-wave velocities) change from t0 to t2.

Between-group difference in white and grey matter volumes and hippocampal brain volume change from t0 to t2.

Between-group difference in brain white matter microstructural integrity change from t0 to t2.

Between-group difference in resting state functional brain activity change from t0 to t2.

Between-group difference in cognitive function change (global composite score) from t0 to t2.

Exploratory outcomes:

Between-group difference in all primary and secondary outcomes from t0 to t1, t2 and t3.

Between-group difference in mood, fatigue and quality of life (QoL) measures from t0 to t1, t2 and t3.

Between-group difference in cerebrovascular function change from t0 to t2 and t3.

Between-group difference in blood biomarkers of neurodegeneration, neurogenesis, metabolism, inflammation, blood lipids and vascular health change from t0 to t1, t2 and t3.

Between-group difference in muscle biomarkers of neuroprotection change from t0 to t2.

Between-group difference in muscle mitochondrial biomarkers (eg, respiration and biogenesis) changes from t0 to t2.

APOE ɛ4 allele status and its effect on training-induced changes in all other outcomes.

Incremental exercise tests

Participants will perform a customised (desired time limit of 10 min) incremental exercise test to exhaustion, as previously described by our labs,61 on six separate occasions (familiarisation, t0, t1 – 2 weeks, t1+2 weeks, t2 and t3). These tests will serve to determine (V̇O2peak; primary outcome) and peak aerobic power (Wmax). Participants will be instructed to exercise until exhaustion, and the test will be stopped if participants reach volitional fatigue (ie, wishes to stop) and/or cannot maintain a cadence of between 60 and 70 rpm (ie, drops below 55 rpm). All incremental tests will be performed under the supervision of an accredited exercise physiologist/scientist, and will be immediately stopped in participants who experience dizziness/chest pain/severe shortness of breath/or any other pain related to (or caused by) exercise; abnormally high heart rates relative to exercise-intensity; signs of metabolic or cardio­respiratory distress; sweating responses that are inappropriate to the environmental conditions in the laboratory; and/or a systolic blood pressure of 225 mm Hg. Oxygen uptake (V̇O2) will be assessed continually during the test using a gas analyser (Quark Cardiopulmonary Exercise Testing, Cosmed, Italy). In addition, heart rate (Polar Electro Oy, Kempele, Finland) and ratings of perceived exertion (RPE; Borg scale)62 will be monitored and recorded every minute. The power at which participants cease exercise will be classified as their Wmax, which will subsequently be used for the determination of exercise intensity during the training intervention (see the ‘Exercise training interventions’ section). The tests performed during the training intervention period (ie, t1 – 2 weeks, t1+2 weeks) will be used to ensure a continual progression of the training stimulus; at these time points, the incremental exercise test will be performed immediately prior to the first training session of weeks 5 and 9, thus avoiding an additional testing day in those weeks. All incremental tests will be performed on an electronically braked cycle ergometer (Excalibur Sport, Lode, Groningen, The Netherlands).

MRI

Brain imaging will be performed using a whole-body Siemens MAGNETOM 7T Plus Ultra-High Field MRI Scanner (Siemens Healthcare, Erlangen, Germany) with a combined 8-channel transmit and 32-channel receive head coil (Nova Medical, Wilmington, Massachusetts, USA). Each scan session will take approximately 60 min including the following sequences: anatomical imaging using three-dimensional T1-weighted sequence with quantitative susceptibility mapping (MEMP2RAGE), pulsed arterial-spin labelling, MR angiogram, diffusion-weighted imaging using a simultaneous multi-slice 2D spin-echo echo-planar imaging (EPI) sequence,63 two-dimensional fluid-attenuated inversion recovery scan and resting-state functional MRI using multiband multislice gradient echo EPI sequence. These scans will be performed immediately after the cognitive function tests at t0, t1, t2 and t3. The MRI protocol can be viewed in online supplemental appendix A.

Cognitive function tests

Cognitive function will be assessed by a battery of neuropsychological tests, administered by trained research personnel who will not be involved in providing the exercise intervention. The test battery is designed to assess global cognition in addition to specific cognitive domains outlined in table 3. These domains were chosen to align with our group’s previous work investigating the longitudinal effects of cerebrovascular disease (eg, stroke) or risk factors on cognitive functioning,64 65 and the effects of cardiovascular exercise on brain and cognitive health in individuals following stroke.66 They also align with recommendations for harmonising assessment of vascular cognitive impairment,67 and have demonstrated sensitivity to the effects of exercise and cardiorespiratory fitness in healthy adults with and without risk of cognitive impairment or dementia.68–70

Table 3

Neuropsychology test battery and questionnaires

Testing will be performed in a quiet and private room, with participants sitting at a desk and the assessor across the other side of the desk. In accordance with the Post Ischaemic Stroke Cardiovascular Exercise Study (PISCES),66 and the Cognition and Neocortical Volume After Stroke (CANVAS)64 study, scores on individual cognitive tests will be calculated in accordance with published normative data, and performance in each cognitive domain will be indicated by (standardised) composite scores from the tests assessing each domain. All cognitive tests will be completed at t0, t2 and t3. Only the CogState computerised tests will be performed at t1, as these tests have demonstrated reliability and stability over multiple short test–retest intervals.71

Mental health, fatigue and QoL questionnaires

The Generalised Anxiety Disorder-7,72 the Patient Health Questionnaire-9,73 the Depression, Anxiety and Stress Scale 21,74 the Short-Form 36 Questionnaire,75 the Fatigue Assessment Scale76 and the Assessment Quality of Life (AQoL)77 questionnaires will be administered to assess general mental health, fatigue and QoL (table 3). These tests will be performed at the same time as the cognitive function tests at t0, t1, t2 and t3.

Cerebrovascular function

Cerebrovascular function will be assessed by measuring cerebral blood velocity of the middle cerebral artery (MCAv) using transcranial Doppler ultrasound (Multigon, Neurovision, Elmsford, New York, USA). After the collection of baseline resting data, MCAv will be measured during three testing conditions designed to challenge cerebrovascular responsiveness. These testing conditions include (in this order) neurovascular coupling (NVC; MCAv response to visual stimulation), dynamic cerebral autoregulation (dCA; MCAv response to changes in blood pressure) and CVR to hypercapnia (MCAv response to increased blood carbon dioxide), as described below. These tests will be conducted on the same day, with 10 min of passive rest between tests, at t0, t2 and t3. In addition to MCAv, heart rate, beat-by-beat blood pressure and end tidal carbon dioxide concentration (PETCO2) will be monitored during all of the cerebrovascular function tests, as previously described.46 Participants will be required to have abstained from moderate/vigorous physical activity, caffeine and food, for at least 6 hour prior to these testing sessions.

At the start of the testing session, the right MCAv signal will be identified and tested using a 2 MHz probe over the right temporal window. Heart rate will be measured continuously using a three-electrode ECG attached to the chest (Bio-Amp, ADInstruments, Bella Vista, New South Wales, Australia). Blood pressure will be measured continuously at the left middle finger using photophlethysmography (Human NIBP, AD Instruments), ensuring the left forearm is supported at the level of the heart using a sling. Finger blood pressure will be exported to generate beat-by-beat continuous mean arterial pressure (MAP). Expired gases will also be collected during the entire testing session, and continuously sampled (PowerLab, AD Instruments) for the determination of end-tidal carbon dioxide (PETCO2). MCAv, heart rate, MAP and PETCO2 will be sampled at 1000 Hz and stored (LabChart Pro, V.8 and PowerLab; ADInstruments).

NVC will be measured during repeated trials of a cognitive task (a reading exercise), as previously described.78 Response times to these tests, and the magnitude of change in MCAv, will provide indices of brain neurovascular function. dCA will measure during repeated sit-to-stand manuvers (SSMs), as previously described.79 Participants will perform 15 SSMs, measured as a 45° flexion of the knee (ie, shallow depth), at a frequency of 0.05 Hz (10 s standing, 10 s squatting), during which MCAv will be recorded. MCAv will also be measured during 90 s periods of standing quietly before and the SSM protocol. During SSMS, a chair will be set at the correct height behind the participant to guide the depth of each squat, and they will be instructed to not put any weight on the chair. Participants will also be asked to avoid a Valsalva‐like manoeuvre (foreceful exhalation with closed mouth and nose) when in the squat positions. CVR will be measured during rebreathing of two different concentrations of carbon dioxide. Following a 5 min baseline collection period (ie, room air; 0.04% carbon dioxide, 21% oxygen and balanced nitrogen), the face mask will be connected to a Douglas bag containing 3% carbon dioxide, 21% oxygen and balanced nitrogen (4 min), and then another Douglas bag containing 6% carbon dioxide, 21% oxygen and balanced nitrogen (4 min).

Blood sampling and biochemical analysis

Venous blood samples (~20 mL each) will be taken from the participant’s antecubital vein via venepuncture at six time points; before and after the first training session, before the training session at t1, before and after the last training session and before the session at t3. Approximately 15 mL of this sample will be split into EDTA and SST blood collection tubes, and the resultant plasma and serum will be stored in a −80°C freezer for future analyses (eg, systemic biomarkers of neurodegeneration, neurogenesis, metabolism, inflammation, blood lipids and vascular health). The resultant ~5 mL of whole blood will be split into an Eppendorf tube (~2 mL) and a Tempus Blood RNA tube (~3 mL) (Applied Biosystems, USA), and stored at −20°C for future DNA/RNA extraction and analysis. Specifically, APOE ε4 genotype will be assessed using a Taqman single-nucleotide polymorphism allelic discrimination assay. Blood lactate will also be measured before and after the first and last training sessions to help characterise the metabolic load of exercise.

Muscle biopsies

A percutaneous muscle biopsy performed with a Bergstrom needle modified with the addition of suction will be used to obtain muscle samples of ~100 to 150 mg from the vastus lateralis muscle of the quadriceps. Muscle samples will be analysed for biomarkers of neuroprotection (eg, myokine/exerkine pathways), as well as biomarkers of mitochondrial regulation (eg, mitochondrial biogenesis and mitochondrial respiratory function). Participants can elect to have none or two muscle biopsies throughout the entire study, and their choice will have no bearing on eligibility to participate in the study. If permission is given, participants will have one muscle biopsy at t0 and another biopsy at t2. Participants will be given the option of choosing which leg the muscle sample is taken from at t0, and the same leg will be used for the t2 biopsy.

Anthropometry assessment

Participant height will be measured while standing barefoot using a generic wall-mounted stature/height measuring tape, and body weight will be measured on a standard scale (PW-200-FG Patient Scale, A&D Weighing, Australia). Participants will be asked to stand wearing just light clothes.

Exercise training interventions

All training interventions will consist of 12 weeks of supervised, work-matched cycling training, performed three times per week on an electromagnetically braked cycle ergometer (Velotron, RacerMate, Seattle, USA). The training programme has been designed to ensure participants achieve the Australian Physical Activity Guidelines for Adults (ie, 150 min of moderate-intensity aerobic physical activity per week).80 Training sessions for the MICT condition will consist of 36–48 min of continuous cycling, performed at an intensity 5 Watts above the power achieved at the ventilatory threshold during the incremental exercise test (~ 60% Wmax), which will be determined as described previously.81 This intensity was chosen as the ventilatory threshold represents the point at which cerebral blood flow has been reported to plateau82 or progressively decrease83 with further increases in exercise intensity, during continuous exercise. Training sessions for the HIIT condition will consist of between four and seven 4 min intervals, interspersed with 3 min of recovery. Interval duration (HIIT) will be set at 4 min in an attempt to maximise shear stress on cerebral vasculature during exercise (and recovery), as acute increases in cerebral blood flow are reported to peak ~3–4 min into an exercise bout.46 84 The intensity prescribed for the HIIT group will be the power achieved at the respiratory compensation point (~ 90% Wmax).81 This intensity was chosen as it represents an intensity with superior cardiovascular health benefits as compared with moderate-intensity training,85 86 without compromising on potential adherence issues. Considering the risks associated with blunted CVR in this population cohort,87 participants in the HIIT group will be required to gradually increase exercise intensity in the last 30 s of each recovery period to ‘prime and prepare’ the cerebrovasculature.88 Each training session will be preceded by 5 min (MICT) or 10 min (HIIT) warm-up at 60% Wmax, which is designed to ensure two groups are work-matched. To ensure adequate training progression, and in the absence of dose-limiting events (eg, a participant is unable to complete the session or an adverse event occurs during the session) training intensity will be increased by 2.5% each week in both groups. In addition, Wmax will be reassessed every 4 weeks during the training period (ie, t1 – 2 weeks, t1+2 weeks) to recalibrate the relative intensity. If a participant experiences a dose-limiting event during training, predefined criteria are in place to modify exercise training to promote adherence and continued participation (eg, regressing the training intensity to the previous week’s intensity). Make-up sessions will be offered to participants who miss training sessions (eg, due to illness or injury) to improve adherence; however, participants will be unenrolled from the study if they are unable to train for two successive weeks (due to the likelihood of detraining), or are unable to achieve a minimum of 30 training sessions over the 12-week training intervention period. To characterise and monitor exercise training load, heart rate (RS800sd; Polar Electro Oy, Kempele, Finland) will be measured and recorded throughout all training sessions for all participants. Additionally, participants will be asked to subjectively rate the perceived exertion of each training session using the session-RPE (sRPE) scale,89 which is a valid and reliable tool for training load monitoring.90 Participants will also be encouraged to maintain their habitual physical activity outside of the training intervention.

Data management

Consistent with the International Council for Harmonisation Good Clinical Practice guidelines, the investigator site file will be located on Victoria University’s internal research drive, an enterprise-grade and secure location for data storage during research and long-term retention. Participant data will be collected in a deidentifiable manner (ie, assigned a study number), and stored on REDCap, a secure web platform for managing online research databases. REDCap user rights will be overseen by the principal investigator (JRB), and assessors will only have access to the data they are unblinded to (eg, assessors conducting the cognitive testing, questionnaires and MRI analysis will have no access to the exercise data collected at Victoria University and vice versa). Personal information (eg, consent forms, risk factor questionnaire) that is collected during recruitment will be stored in a locked cabinet on site at Victoria University (accessible by JRB and NZ only), ensuring confidentiality before, during and after the trial.