Effects of 12-week dairy-derived nutritional supplementation combined with physical activity on health and physical function in healthy middle aged women after a 2-week period of reduced activity

INTERVENTION: Participants will be randomised into 2 treatment groups and progress through 2 phases (Phase 1 = 2 wk reduced activity; Phase 2 = 12 wk exercise + supplementation intervention). In Phase 1 the participants will aim to reduce their physical activity to below 1500 steps per day. During this period the participants will: (1) spend the majority of their day engaged in sedentary behaviour (i.e. activities with energy expenditure close to the resting level of 1.0‐1.5 metabolic equivalent units (METs), including activities such as sleeping, sitting, lying down, watching television, and other forms of screen‐based entertainment, (2) spend less than 90 min per day engaged in light activity (energy expenditure of 1.6‐2.9 METs, including activities such as slow walking, cooking food and washing dishes), and (3) refrain from moderate and high intensity activity. Steps will be recorded each day using a pedometer to assess compliance. To determine energy expenditure across this period an accelerometer will also be worn daily (attached to the waist belt), and 7‐day IPAQ questionnaires will be completed. A 3‐day weighed diet record will also be completed, and testing will be completed, before and after this 2‐week phase of reduced activity. In Phase 2, commencing immediately after Phase 1, the participants will begin supplementation with a fortified milk powder (FMP) or a placebo (PLA), and complete a 12‐week structured physical activity (i.e. healthy lifestyle) programme that will include four training sessions a week: two university‐based (strength, balance and flexibility training done in a group/social environment) sessions completed in small groups of up to 8 participants as well as two home‐based (calisthenics, balance and flexibility training) sessions performed individually. The exercise programme will include elements of traditional strength and flexibility training programmes alongside aspects taken from yoga, Pilates, body balance and other popular forms of exercise. For the university‐based gymnasium exercise program, the participants will complete 50 min of broad‐based exercise training including muscle stretching, balance, muscle strength and physical fitness/endurance exercises. The sessions will commence with mobility and muscle stretching exercises (15 min). Fitness and muscle strength training will then follow, and the session will conclude with a short (5 min) stretch and warm‐down period. Strengthening exercises (20 min) focusing on a holistic approach, but targeting the lower limb, will be incorporated into all sessions; examples include body‐weight squat and lunge movements, although leg extension exercises performed on a machine will be incorporated. The strengthening exercises will be completed as a circuit format with 45 seconds work to 15 seconds of rest. Within those 45 seconds, 10‐15 reps will be performed at an intensity of 14‐15/20 RPE. For the fitness/endurance component (8 min per session), participants will perform stair climbing (4 and 3 min bouts with 1 min rest) or cycling, rowing and/or walking/running at a ‘moderate intensity’ in intervals with a 1:3 high‐ vs. low‐intensity work ratio (e.g. cycle 15 s on level 12 then 45 s at level 4). All trainers will have a minimum of a degree level qualification in Exercise and Sport Science. Phase 2 treatment arms: 2. Placebo, 30 g dose, 2 x per day; total dose 60 g/day [PLA] ‐ see 'Comparator / Control CONDITION: Bone health Mental health Physical function Physical health PRIMARY OUTCOME: Ascending ; ; SECONDARY OUTCOME: Ankle joint range of motion (flexibility) ; ; ; Balance (simple) ; Blood‐based biomarkers of bone health ; Bone structure and strength ‐ pQCT ; Descending stair climb ; ; Dynamic balance and agility At home the participants will perform a basic 20 ‐ 30 min session that mirrors the movements and exercises performed in the gymnasium but which can be achieved without supervision or equipment. A reference sheet with photographic explanations will also be supplied as further reference. They will begin with a 10 minute warm up of active range of motion exercises, and then perform 10 minutes bodyweight‐resistance exercises. These resistance exercises cover 4 different movements. One set of each is to be completed to be completed to as many repetitions as possible. The intensity of these body weight exercises will therefore begin very light and then peak with a final repetition around 17‐20/RPE. During Phase 2, the participants will also complete at least 3000 steps per day on ‘non‐exercise’ days. In this phase of the project we will determine whether daily supplementation with a dairy‐derived nutritional supplement provides greater health, mobility/physical function and vitality outcomes than physical activity alone, and thus whether this nutritional strategy might be adopted in 40 – 60 year‐old women seeking to improve their health and well‐being. The participants will be tested after 6 and 12 weeks of the programme. 1. Fortified Milk Powder, 30 g dose, 2 x per day; total dose 60 g/day [FMP]. The FMP supplement is largely comprised of milk powder (skim and whole), vitamins, minerals, complex milk lipids, maltodextrin and a small amount of hydrolysed fish collagen. It also contains low levels of Vitamins B12, B6, E and C. ; The participant will be instructed to walk or run as quickly as possible up 11 stairs each 16 cm high, without missing a step. The participant will start at the base of the stairs with both feet together and then ascend the stairs on the ‘go’ command of the experimenter. Timing will commence when the participant makes their first movement and will stop when both feet are placed on the final step. Two trials will be recorded to the nearest 0.01, and reported to the nearest 0.1 s, with 1 min of rest between (including slow stair descent). ; The fastest stair ascent time will be taken as stair climb performance. Additionally, stair climb power for the 11‐step test will be measured using the equation: Power (watts) = 9.81 × body mass (kg) × vertical height (m) / time (s). ; Passive ankle dorsiflexion rotations will then be completed starting at 20 degrees plantar flexion (0 degrees = neutral position) and stopping when the participants press a stop button, which immediately releases the footplate and moves the ankle back to the start position. A slow joint angular speed of 5 degrees·s‐1 will be used to ensure that variations in muscle‐tendon viscosity and Ia (stretch)‐mediated mechanical responses will not influence the mechanical response to the stretch, and the participant’s eyes will be closed to prevent visual feedback from influencing the stretch amplitude. Two trials will be performed with a 30‐s inter‐trial rest period, and the greatest dorsiflexion angle attained in either trial will be taken as the maximum ROM. ; During post‐hoc analysis, the maximum ROM as well as the musculo‐articular stiffness (calculated as the ratio of the change in joint moment to the change in joint angle at successive 10 degree intervals) will be determined and used for analysis. ; Maximum ankle joint range of motion and passive stiffness (resistance to muscle stretch) in the calf muscles will be assessed during a maximal dorsiflexion joint rotation on an isokinetic dynamometer. The participant will be secured in a seated position in the chair of the isokinetic dynamometer with the knee angle of the right leg at 0 degrees (i.e. anatomical position). The participant be seated (hip angle at 85 degrees; 0 degrees = full extension) with the foot (unshod) firmly strapped to the ankle dorsi/plantar flexion footplate of the dynamometer and the lateral malleolus aligned with the dynamometer’s axis of rotation. The chair will then be positioned such that a small knee extension movement is required to straighten the knee, which will slightly deform the chair and dynamometer shaft which will then minimise heel lift from the footplate during ROM testing. Additional strapping across the foot, waist and chest will be used to minimise movement without impacting on dorsiflexion ROM. ; Single‐leg balance testing will be done with the participant standing for 30 s unshod on one leg in the centre of a force platform. The non‐weight bearing foot will be raised to ankle height, and the experimenter will remain within reach to provide support in case the participant loses their balance. The test will be done in two conditions, eyes open followed by eyes closed, and balance performance of both legs will be examined. If the participant fails to complete the first trial in each condition, they will be given 2 additional attempts to complete the full 30 s. There will be a 5‐s inter‐trial rest and the order of dominant and non‐dominant leg will be randomised between participants but kept constant at successive test sessions. Both the balance time and displacement of the centre of pressure from the force plate (COP variability) will be recorded. ; Venous blood samples will be obtained by trained phlebotomists using sterile Vacutainers (plasma separator and EDTA buffered tubes) and needle between 06:00 and 09:00. The participants will report to the laboratory after an overnight fast of at least 8 hours (excluding water) for a blood draw from the antecubital vein. Blood collected by EDTA Vacutainer will be kept on ice, centrifuged, and then separated into 500 uL aliquots of plasma (stored in 1.5 mL sterile screw‐top tubes) and stored upright at ‐80 degrees C until analysis (<2 h between blood sampling and plasma freezing processes). Samples will be transported on dry ice for analyses at Massey University, New Zealand (or laboratories appointed by them), and analysed for bone markers including CTX‐I, PINP, PTH, Sclerostin, OPG and Vit D. Blood‐based health biomarkers ; Venous blood samples will be obtained by trained phlebotomists using sterile Vacutainers (plasma separator and EDTA buffered tubes) and needle between 06:00 and 09:00. The time of sampling will be held constant (+/‐30 min) between occasions because of the circadian variability of bone marker concentrations. The participants will report to the laboratory after an overnight fast of at least 8 hours (excluding water) for a blood draw from the antecubital vein. CRP, hsCRP, Glu, LDH, Cholesterol, triglyceride, cLDL, HDL, E2, FSH, Progesterone and FBP concentrations will be determined using standard procedures by the pathology laboratory (Pathwest, Australia). Body composition ‐ DEXA ; Dual energy x‐ray absorptiometry (DEXA) will provide a non‐invasive measure of body composition, including an indirect assessment of fat‐free mass (FFM). Participants will attend the lab well hydrated but refrain from water/fluid ingestion for 1 h prior to the scan and void their bladder (this may be used to obtain a spot urine sample a several time points) prior to scanning. Participants will be positioned supine with legs extended and arms resting by their sides on the DEXA table and asked to lie still during the scan. A single whole‐body DEXA scan will be used, and body composition (which is a composite of total body fat, fat‐free soft tissue and bone mineral content) will be determined. FFM (proxy for skeletal muscle mass) will be calculated as the sum of fat‐free soft tissue and bone mineral content. The total radiation exposure per DEXA scan is 0.0002 mSv which is comparable to the background radiation exposure over the course of a normal day. ; Bone structure and strength of the lower leg will be determined by peripheral quantitative computed tomography (pQCT; XCT 3000; Stratec Medizintechnik, Pforzheim, Germany) of the right leg at 4% (distal) and 66% (proximal) of the lower leg length from the distal end of the tibia, as located by a scout scan. The participant will be seated with their right leg in the bore of the scanner and the left leg supported outside the bore. The bore position will be adjusted so that the scan is completed at the required site, and the scanning commenced. The composite measures of bone mineral density and content, along with stress‐strain index (SSI) will be determined using the Stratec software post‐hoc to assess bone structure and strength. ; The participant will be instructed to walk or run as quickly as possible down the 11 stairs, each 16 cm high, without missing a step, with both feet starting on the top (11th) step. The time will be recorded to the nearest 0.01 s from when the participant makes their first movement to when both feet are placed on the final (bottom) step, and reported to the nearest 0.1 s. ; The fastest stair descent time will be taken as stair climb performance. Additionally, stair climb power, for the timed 11‐step descent will be measured using the equation: Power (watts) = 9.81 × body mass (kg) × vertical height (m) / time (s). ; Hand grip strength will be obtained on the left and right hands. The participant will stand with arms by their sides and elbows slightly bent, and squeeze the dynamometer with as much force as possible. Three 3 s trials will be completed with 15 s rest between, and the maximum value used for analysis ; Data will be acquired at a 1000‐Hz sampling frequency and filtered with a 14 Hz low‐pass, 4th order zero‐lag Butterworth filter; the highest reading will be taken as the maximum isometric knee extensor strength. Muscle size (ultrasonography) ; Plantar flexor (gastrocnemius) muscle thickness will be obtained from a single scan taken in the middle of the muscle belly at 66% of the distance from the distal end of the tibia to the lateral epicondyle. ; The testing sites will be measured and marked in the middle of the vastus lateralis muscle belly at 50% of the distance along a line from the greater trochanter to the lateral epicondy, and in the middle of rectus femoris at 50% of the distance along a line from the anterior superior iliac spine to the superior border of the patella. ; The timed up and go test is designed to test a participant’s dynamic balance and agility (i.e. functional mobility). The participant will start the test sitting in a ‘standard’ chair that is placed firmly against a wall, with their back straight, feet flat on the floor with one slightly in front of the other, hands on thighs, and torso slightly leaning forward. On the ‘go’ signal, the stopwatch will be started by the researcher and the participant will leave the chair, walk around one side of a cone placed 3 m from the chair, and then sit back down on the chair as quickly and safely as possible. Timing is stopped when the participants sits clearly on the chair in an upright position with their hands on their thighs. After familiarisation, one properly‐executed trial will be allowed with time recorded to the nearest 0.01 and reported to the nearest 0.1 s. ; Gait speed ; Self‐paced walking speed ; A 6‐m distance will be clearly marked on a Zeno walkway (containing 1 cm2 pressure sensors) that is placed on a flat, hard floor, with timing gates placed at the 0 and 6‐m points (alternatively, a stop watch can be used). The participant will stand 30 cm behind the start line to prevent triggering of the timing gate, with both feet together. When the participant is ready, they will walk “at your usual walking speed, just as if you were walking down the street to go to the shops”. Using a stop watch, the time to walk 6 m will be recorded and stopped when one foot touches the ground past the 6‐m line. Time will be recorded to the nearest 0.01 s, the test will be repeated after 30 s of rest, and the mean result taken as indicative of self‐paced walking time. Walking speed will then be calculated as distance (6 m)/time (s). ; ; Maximum walking speed ; This test is performed under similar conditions to the self‐paced walking speed test, however the instruction is to “walk as fast as you possibly can past the 6‐m line on my command” and the participant will begin walking from 1 m advanced of the start line to a finish line that is 1 m past the 6 m finish line. Two attempts will be given with a 30‐s rest, and the fastest time and kinematic data from the fastest trial will be recorded. The researcher will watch to ensure that the participant doesn’t run, which is indicated by a bent knee during the ground contact phase and a period of the gait cycle without one foot clearly touching the ground (i.e. no airborne phase is allowed). ; General flexibility (sit‐and‐reach) ; General flexibility will be measured using the sit‐and‐reach test. The participant will sit on the floor with shoes removed with the legs extended together straight out in front of the body. The feet will sit firmly and flat against a sit‐and‐reach box, then instructed to reach forward with their overlapping hands (third digits overlapping) as far as possible. The maximum stretch held for at least 3 s will be recorded. During each attempt, the participant will be instructed to exhale and drop the head forward whilst reaching. The experimenter will ensure the knees remain extended (0 degree knee angle). Three trials will be allowed with the greatest score taken for analysis. Grip strength ; Knee extensor strength ; ; ; ; Muscle thickness will be measured as the greatest observable distance between the superficial and deep aponeuroses of each muscle. Muscle stiffness ; ; ; Queens’s College Step‐up Test (cardiorespiratory fitness) ; Vertical Jump test (lower body power) ; ; INCLUSION CRITERIA: 1. age = 40‐60 y 2. Female 3. BMI (18 ‐35 kg/m2) 4. Sedentary to moderately active 5. Healthy ; Unilateral isometric strength testing will be performed on the right leg. After being seated in the isokinetic dynamometer (Biodex System 4, Chatanooga, USA) with the knee joint centre of rotation aligned with the axis of the dynamometer’s lever arm, the participant will perform 3‐s isometric knee extensor contractions at 50, 70 and 90% of their perceived maximum at a 45 degree joint angle (10 s rest between contractions). They will then perform three maximal 3‐s knee extensions separated by 30 s of passive rest with the instruction to contract “as fast and then as hard as possible”. Trials showing a drop in joint moment >5 Nm at the start of contraction will be discarded (due to the active counter movement) and another trial completed. After a 2‐min passive rest the warm‐up and testing will be repeated with a 90 degree knee joint angle. ; Quadriceps (vastus lateralis and rectus femoris) and calf muscle (gastrocnemius) muscle thickness will be measured using B‐mode ultrasonography, to estimate muscle size. Ultrasound images of the quadriceps will be taken at two sites: (1) on the anterior thigh at 50% of the distance along a line between along the midline between the medial aspect of the anterior superior iliac spine and the middle of the superior border of the patella on the rectus femoris muscle, and (2) in the middle of the vastus lateralis muscle belly at 50% of the distance along a line from the greater trochanter to the lateral epicondyle. The ultrasound probe (60 mm width, 10 MHz scanning frequency, gain = 58 dB) will be placed perpendicular to the skin and oriented horizontally. Two images will be taken at each site and saved for analysis. The images at site 1 will allow measurements of rectus femoris muscle thickness, and the images at site 2 will allow measurements of vastus lateralis. ; Two minutes after the completion of the knee extensor strength tests at each joint angle, muscle stiffness will be measured using the Myoton Pro device. A small amplitude quick‐release mechanical impulse will exerted by the device at a constant force to the tissue layer directly above the muscle. This compressed layer then transmits the impulse to the muscle below, which responds with a damped oscillation response that is recorded by the devices accelerometer. The filtered oscillation data are then used to compute variables that describe the tissue’s viscoelastic properties, including: the natural oscillation frequency (Hz) as an indicator of tissue stiffness (tone or tension); the logarithmic decrement of the natural oscillation (elasticity/damping) and dynamic stiffness (N/m) as indicators of the biomechanical properties; and the mechanical stress relaxation time (ms) and ratio of deformation and relaxation times (Deborah number) as indicators of viscoelastic properties. ; At each site, measurements will be taken during complete rest (relaxed muscle) and whilst holding an isometric contraction at 20% of the maximum torque recorded at test point 1 (i.e. the first testing session), as determined in the preceding maximal isometric test. The device probe will be placed perpendicular to the skin surface and directly on the skin, and slight pressure will be exerted on the underlying soft tissue. Two sets of tests, consisting of 10 beats each (one second apart, 15 ms in duration) will be conducted at each point. If the CV reads above 3% for any measure the test will be discarded and re‐conducted. ; The Queen’s college (McArdle’s) step test provides a valid estimate of VO2max. Whilst wearing comfortable clothing including flat shoes and after familiarisation, the participant will perform 3 min of stepping up and down on a 41.3 cm step at 22 steps per minute. A pulse oximeter will be fitted to the finger whilst the participant stands quietly in front of the step (the participant should be relaxed and heart rate at resting speed), with heart rate (HR) after 10 s. After HR is recorded, a metronome will be started at 88 beats per minute. When ready, the participant will step up with one leg and then up with the other leg, then down with the first leg and down with the other leg, completing one step‐up cycle. The experimenter will make sure that the whole foot lands on the step. The test should be stopped if HR exceeds 85% of age predicted maximum or the participant experiences any other symptoms that indicate the cessation of exercise. The participant steps continually in time with the metronome for 3 min. At test completion, the oximeter will be immediately placed on the finger (if it was removed for the test) and HR read after 5 s. The change in HR from pre‐ to post‐test will be recorded, and VO2max will be estimated using the formula for women: VO2max (ml/kg/min) = 65.81 ‐ (0.1847 x heart rate (bpm)). ; The vertical jump test is designed to test lower body muscular power output. Following warm‐up/familiarisation of two practice trials, the participant will commence the test standing with both feet in the centre of a force platform (Kistler Instrumente, Switzerland; sampling frequency = 1000 Hz) approximately shoulder width apart and the hands placed on the hips. Once the force platform is triggered for data collection, the participant will be asked to jump “as high as possible, then land on both feet on the platform with legs initially extended”. ; The participants will perform the jump by dipping (squatting) to a self‐selected depth and then jumping as high as possible. They will perform both a squat jump, and a counter movement jump. Three attempts of each will be allowed with 30 s between. Jump height will be calculated and used as performance indicators.