Body composition and bone mineral density (BMD) will be assessed using a dual energy x-ray absorptiometry (DXA) scanner (Hologic, Horizon A, software version 5.6.0.4). Total BMD as well as the neck of the femur and lumbar spine BMD will be assessed. Lean body mass and fat mass will also be assessed. The DXA scan will ideally be performed in the morning following an overnight fast by experienced personnel. This will be completed by the AIMSS.

Peripheral quantitative computed tomography (pQCT; Stratec XCT3000, Stratec Medizintechnik GmbH) will be used to quantify muscle and bone mass, density, and adipose infiltration at the nondominant forearm and foreleg [45,46].

Single 2.5-mm transverse scans will be obtained at 4% and 66% of tibial length (measured form the palpable tip of medial malleolus) and 4% and 66% of the radial length (from the radial condyle), with a voxel size of 0.4 mm. All pQCT scans will be acquired and analyzed by an experienced operator, and the device will be calibrated on the scan date using the manufacturer’s phantom. Calf and forearm muscle cross-sectional areas (mm²) and densities (mg/cm³) will be determined using the manufacturer’s algorithms and software (version 6.2). The calf intramuscular adipose tissue cross-sectional area (cm²) will be quantified as previously described [47]. Trabecular and cortical bone densities and structure will be assessed at the relevant regions of interest. All imaging will be performed by an appropriate expert (radiographer).

Beta-isomerized C-terminal telopeptide (a bone resorption marker) and procollagen 1 N-terminal propeptide (a bone formation marker) will be quantified using a Roche Hitachi Cobas e602 immunoassay analyzer, according to the manufacturer’s guidelines. Hormones (PTH), lipids, glucose and insulin, inflammation markers (C-reactive protein and serum IL-6), and potentially other cardiovascular or health markers will be analyzed according to standard hospital procedures.

We will target either candidate gene variants [48,49] or Genome-Wide Association–based variants previously related to skeletal muscle and bone health [50,51]. Genomic DNA will be extracted from residual blood samples from Becton Dickinson (BD) Vacutainer EDTA tubes using the MagSep Blood gDNA kit (0030 451.00, Eppendorf) or GeneJET Genomic Whole Blood DNA Purification Kit (#K0781 Thermo Scientific). Gene variants will be determined using the TaqMan SNP assay (Applied Biosystems, Thermo Fisher Scientific) by QuantStudio 7 Flex (Applied Biosystems, Thermo Fisher Scientific). Genotyping will be replicated in another independent institute, as previously described [52,53], to validate the results.

Grip strength will be measured using a hand dynamometer; a result of <20 kg for women and <30 kg for men will identify low muscle strength [54,55]. A 4 m gait velocity assessment will be performed by using the instrumented walkway, which has an acceleration (GAITRite), and by timing with a stopwatch, and reduced physical function will be determined as <80 cm/second. Both the grip strength and gait velocity thresholds noted are accepted as a measurement of sarcopenia [54,55] and will form the definition in this study.

Participants will perform a one-repetition maximum (1RM) test on a leg press. This will be performed twice, with the first visit serving as familiarization. 1RM is defined as the heaviest weight lifted once with the proper technique and without compensatory movements [56]. Results from this study will guide appropriate prescription for the acute RE session.

LMQ, an estimate of specific force, has been shown to decrease with age and is described as the amount of force a muscle group can produce per unit of muscle mass [57]. We will calculate LMQ as follows:

LMQ = leg strength (kg)/(left leg lean mass [kg] + right leg lean mass [kg]) (1) [58].

Leg strength will be defined as the participants’ 1RM, and leg lean mass will be obtained from the DXA assessment.

Participants will complete a physical performance test (PPT), adapted from Levinger et al [59], and will include 4 functional mobility tasks: (1) a gait velocity assessment (described earlier), (2) timed up and go test, (3) stair climbing power (SCP), and (4) stair descending. All tests will be scored in time (seconds).

The timed up and go test is a simple performance-based assessment that requires minimal equipment, including a standard arm chair (approximately 46 cm), a 3-m walkway with a floor mark, and a stopwatch (time, seconds). It is performed as time (seconds) taken to rise from a seated position, walk 3 m, turn, walk back to the chair, and then sit. The SCP consists of a rapid ascent of 10 stairs and is calculated as follows:

Power = force × velocity (2)[60]

Velocity is calculated as the vertical distance of the stairs divided by the time it takes to ascend the stairs. Force is calculated as the participants’ body weight multiplied by acceleration due to gravity (9.8 m/s²). The stair descent will be the time to safely descend 10 stairs. The rest between ascent and descent will be 45 seconds. Participants will undergo 4 attempts on each task, and the best time will be recorded for each task. The PPT score will be the sum of the fastest times recorded for each test.

Participants will complete a lifestyle behavior and physical activity log. This log has been developed for the purpose of this study and will have questions related to average sleep cycles and normal physical activity levels on weekdays versus weekends (stratified into moderate, hard, and very hard activities). The physical activity component includes consideration for activities of daily living and structured exercise, with examples provided.

A 3-day dietary log will be given to participants on their first visit, to be returned on visit 2 for investigators to analyze normal dietary behaviors. Participants are encouraged to eat normally while they are recording (ie, not to adjust food quantities) and are instructed to complete the dietary log on 2 weekdays and 1 weekend day (consecutively). This log also requests a timed record of physical activity behaviors, including the time and intensity, the time at which food and drinks are ingested, and the time and quantity of medications and supplements.

The Falls Risk for Older People in the Community (FROP-Com) was developed by the National Ageing Research Institute as a modified version of the Falls Risk for Hospitalized Older People for better utility in the community [65]. The FROP-Com is simple, takes only 10-15 minutes to complete, is low cost, requires no equipment, and can be administered by any health professional. It is a comprehensive fall risk assessment, covering 13 risk factors for falls set out in 26 questions with dichotomous or ordinal scoring (from 0 to 3). The overall score is indicative of fall risk, with the total score ranging from 0 to 60, with higher scores indicating greater risk. The tool has demonstrated good reliability and has a moderate capacity to predict falls [65].

The Mini Nutritional Assessment (MNA) is a widely used tool for assessing nutritional status in older adults. It is simple to administer, low cost, and validated, with high sensitivity, specificity, and reliability. The MNA classifies the interviewee as well nourished (score≥24), at risk of malnutrition (score between 17 and 23.5), or malnourished (score<17). The MNA also correlates with clinical assessments and objective measures, such as albumin, BMI, triceps skinfold, caloric intake, and vitamin status, and low scores are related to the incidence of clinical events and mortality [66-69].

The Charlson Comorbidity Index (CCI) is a validated measure of 1-year mortality risk and burden of disease and is used in clinical research to understand the influence of comorbidities and predict outcomes [70-72]. In clinical practice, the CCI assists with the stratification of patients into subgroups based on disease severity to assist with targeted models of care and resource allocation. The CCI includes 17 comorbidities (with 2 subgroups for diabetes and liver disease) that are weighted from 1 to 6 for mortality risk and disease severity. These scores are then tallied to form the total CCI score.

Participants will complete the acute intervention (visit 3, 4, and 5) up to 14 days after completing the screening assessments and will complete the AER, RE, and CON conditions (Textbox 2) in a randomized order (described below). These visits will include blood sampling and optional skeletal muscle biopsies. Participants can elect to have none, 1 (at rest, for a baseline measure), or 4 biopsies (1 at rest for baseline and 1 following each condition). Each testing visit is approximately 3 hours in duration, including the 30-minute intervention (exercise or rest), and visits will be performed approximately 7 days apart, accounting for washout.

All testing visits will be monitored and supervised by accredited exercise physiologists (AEPs), who will follow the structured protocol as dictated for that particular session (AER, RE, and CON). The AEP will also monitor signs and symptoms in response to exercise training and will record Borg RPE, blood pressure, and heart rate at frequent time points. Any adverse signs and symptoms will be documented, including feelings of fatigue, soreness, light-headedness, and any injuries. Blood sampling and intravenous cannulation will be performed by personnel who are experienced in the technique, and muscle biopsy will be performed by an experienced medical physician.

For testing visits 3, 4, and 5, participants will arrive at the laboratory between 7 AM and 8 AM following an overnight fast and with abstinence from exercise or reduced general activity (ie, heavy to moderate activities of daily living) in the preceding 24 hours and for all follow-up sessions. These sampling procedures will be followed at all visits to account for circadian or diurnal rhythms [76]. Participants may be requested to abstain from particular medications (eg, aspirin), as advised by the medical doctor, if electing for a muscle biopsy.

To assist with adherence to study protocols, participants will be monitored via regular communication with the study coordinator on the days preceding each study visit. As a general consideration for participation in this study, participants will be encouraged not to alter their current physical activity levels, exercise habits, or dietary intakes for the entirety of the study. Participants are asked to report whether there have been any alterations in medications throughout the study, as we request that all medication interventions are stable for at least more than three months.

On arrival and following supine rest (approximately 15 minutes), a cannula will be inserted into the antecubital vein, and a baseline (resting) blood sample (40 mL; biopsy, if consented) will be obtained. These baseline (resting) samples will be obtained on the first visit only, serving as the baseline for all other visits thereafter. Four additional blood samples following the 30-minute acute intervention will be collected immediately after the intervention (0-minute time point) and at 30, 60, and 120 minutes postintervention (total of 100 mL) to observe changes in tOC, ucOC, COP, and other measures. If elected, a postintervention biopsy will be conducted at the 60 minutes time point. At all time points, blood samples will be collected into EDTA and serum-separating tubes vacutainers for the appropriate collection of serum or plasma. Following 10-minute clotting time, samples will be centrifuged for 10 minutes at 4° C and immediately transferred to long-term storage at −80° C in 2 mL aliquots for later analysis.

The primary outcome for this study is the peak change in circulating levels of ucOC from baseline compared with postacute exercise blood sampling time points (0, 30, 60, and 120 minutes) following the 3 acute interventions (AER, RE, and CON) between the low muscle function and high muscle function groups.

The secondary outcomes for this study are (1) the difference in protein content related to atrophic and hypertrophic protein signaling at baseline between the low muscle function and high muscle function groups and (2) the difference in protein signaling (protein content) from baseline and compared with the postmuscle sampling timepoint (60 minutes) following the 3 acute interventions (AER, RE, and CON) between the low muscle function and high muscle function groups.

Serum tOC will be analyzed as described previously [9,10,19,39]. In brief, tOC will be measured using an automated immunoassay (Elecsys 170; Roche Diagnostics). Serum ucOC will be measured by the same immunoassay after absorption of cOC on 5 mg/mL hydroxyl-apatite slurry, following the method described by Gundberg et al [77].

COP cell analysis will be performed as described previously [78,79]. In brief, peripheral blood samples (20 mL) will be collected (EDTA tubes) and processed for Ficoll-based gradient separation, and approximately 5×10⁶ peripheral blood mononuclear cells (PBMCs) will be obtained. Approximately 1×10⁶ PBMCs will be resuspended in fluorescence activated cell sorting (FACS) buffer, followed by a 10-minute blocking with fragment crystallizable receptor blocking reagent (BD Biosciences). Staining will then be performed with a viability marker (30 minutes), followed by washing (×2) with phosphate buffered saline (containing 5% fetal calf serum). Cells will be incubated with mouse antihuman CD45-Pacific Blue, CD3-PerCP, and CD19-APC (40 minutes). Staining of intracellular components will be permeabilized with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s instructions, followed by incubation with mouse antihuman osteocalcin-phycoerytrhin at 4 °C (40 minutes), and then washed with Perm or wash buffer (×2).

Cells will be analyzed using a BD FACS Canto II. FACS DiVa software will be used to analyze 50,000 total events for each sample and for the fluorescence minus one (FMO) controls. A total of 3 lasers and 8 different photomultiplier tube (PMT) channels will be used for the 6-color staining panel. Two FMOs will contain fluorochromes, except for the one to be controlled for. Compensation beads will be used to set the compensation controls for each fluorochrome. The PMT voltage values for fluorochromes will be set for each cell type based on the compensation controls. Doublet discrimination will be applied, and viability will be assessed by negative staining using the Live/Dead stain. Offline analysis will be performed using Flow Jo analytical software (Treestar).

Cells will be gated for size, shape, and granularity using forward and side scatter parameters, as previously described by our group [78,79]. Briefly, serial gating steps will be applied to quantitate cellular populations. First, dead cells will be excluded, and then, a region will be set to encompass lymphocyte-, monocyte-, and granulocyte-enriched areas, followed by doublet discrimination, T cell (CD3) and B cell (CD19) elimination. For COP cells, after gating on live single mononuclear cells (on forward and side scatter plots), the CD45 and osteocalcin double positive cells will be calculated. Cutoff points to assign antigen positivity will be performed against matching FMO controls. The use of FMO significantly increases the sensitivity and specificity of analysis, as they effectively minimize the effect of nonspecific antibody binding and cell-specific autofluorescence. The gating quantification will be performed twice for the accurate quantification of the percentage of COP cells.

If elected, the muscle samples (1 or 4) will be taken from the vastus lateralis (approximately 150 mg) under local anesthesia (xylocaine 1%) by percutaneous needle biopsy technique, modified to include suction [80]. Excised tissue will be snap frozen in liquid nitrogen and stored at −80° C for later analysis. Proteins involved in muscle degradation and hypertrophy (ie, anabolic and catabolic pathways; ubiquitin-proteasome, autophagy-lysosome, and caspase-3–mediated proteolytic pathways) as well as glucose uptake will be assessed, as described previously [81-84].

All muscle samples (baseline and postintervention samples) will be used to analyze the content and activation of signaling proteins involved in muscle degradation and hypertrophy by using western blotting, as described previously [84-86]. Western blotting is a method commonly used to detect and analyze the abundance and posttranslational modifications (such as phosphorylation) of proteins. Briefly, muscle samples will be homogenized in a radioimmunoprecipitation assay buffer using a TissueLyzer (QIAGEN). Then, proteins in the lysate will be separated based on protein molecular weight via gel electrophoresis. Proteins will be subsequently transferred to a polyvinylidene fluoride membrane where specific proteins can be probed using specific antibodies. Finally, signals generated through electrogenerated chemiluminescence will be detected and analyzed using ChemiDoc Imaging Systems (Bio-Rad Laboratories).

A portion of the muscle obtained at rest (baseline) will be used for cell culture for future molecular analyses [87]. This will be established according to the method described by Blau and Webster [88] and by Gaster et al [89] and previously detailed by McAinch et al [90]. Briefly, muscle samples (50-100 mg) will be washed, minced, and enzymatically dissociated with trypsin. Cells will be cultured in a coated flask with extracellular matrix, and the growth medium (α-minimal essential medium [α-MEM]+10% fetal bovine serum+0.5% penicillin-streptomycin+0.5% antifungal) will be changed every other day until they reach 80% confluence. Then, satellite cells will be selected using CD56+ magnetic microbeads (Miltenyi Biotec) and transferred to bigger flasks coated with extracellular matrix to increase cell number (up to 4 passages). Once the cells reach 80% confluence, they will be differentiated using a differentiation medium (α-MEM+2% horse serum+0.5% penicillin-streptomycin+0.5% antifungal) for 5-6 days. Before the experimental treatment, cells will be starved for 1 hour in a serum-free medium (α-MEM+0.5% penicillin-streptomycin+0.5% antifungal).

In vitro treatment with ucOC at concentrations of 0 ng/mL, 30 ng/mL, and 100 ng/mL in serum-free medium for 60 minutes and 24 hours in the presence or absence of insulin (100 nM for the last 15 minutes) for the determination of glucose uptake (2-deoxy-D-[3H] glucose) and western blotting will be performed. Analysis of targeted proteins (described above) will be performed as described previously [39,91]. The dose response is important because the physiological effect of ucOC in muscles from mice and humans may be different, and the concentrations used are all physiologically relevant.

Details of the procedures for data management have been reviewed and approved by the MH Human Research Ethics Committee and can be located via study reference, 2017/08. A trial management group (TMG) has been formed to manage potential risks and for structured oversight of the trial [93]. The type of oversight that this TMG will provide includes regular meetings to review individual safety reports and data relating to quality, protocol adherence, and participant retention rates. The TMG committee will include the principal investigator; individuals responsible for the daily running of the trial, including the trial coordinator; and an appointed independent member.

All electronic data will be stored on password-protected computers. Hard copies of any data will be kept in a locked filing cabinet in a secure office. All data collection tools and questionnaire data will be deidentified.

All adverse events associated with the study, or occurring during study participation, will be recorded. All adverse events will be reported to the TMG and ethics committees (MH and Victoria University) with strategies to reduce the risk for future events. The ethics committees have the power to pause or even stop the research in the case of a severe adverse event. The study personnel will monitor the clinical signs and symptoms of dyspnea, shortness of breath, nausea, faint-headedness and light-headedness, signs of inflammation, or infection throughout the study period.

The TMG will meet and run an internal audit of the trial at regular intervals annually. The principal investigator, IL, is responsible for the overall conduct and preservation of the integrity of this trial and has extensive experience as a lead research investigator in numerous human clinical trials.