Minimizing sedentary behavior (without increasing medium-to-vigorous exercise) associated functional improvement in older women is somewhat dependent on a measurable increase in muscle size

Results

Descriptive characteristics of participants at baseline

The 34 older women were matched at baseline for all outcome variables of interest (Table 1). Briefly, there was no statistically significant difference between the three groups at study onset for either GM, GL, or VL regional anatomical cross sectional area (CSA), total muscle volumes, or fascicle length (Lf). Furthermore no baseline differences were observed for Achilles Tendon average or regional CSA, nor any of the segmental DEXA-derived body composition outcome variables (Table 2). Only VL fascicle pennation angle (FPA) (p≤0.001) and VL physiological cross-sectional area (PCSA) (p=0.005) exhibted signficant differences between groups at baseline.

Table 1. Baseline characterstics of the study sample.

		SBF (n=13)	LIPA (n=13)	Control (n=8)	Whole sample (n=34)
Age (years)		74 ± 5	74 ± 6	70 ± 3	73 ± 5
Height (m)		1.59 ± 0.07	1.61 ± 0.07	1.58 ± 0.1	1.60 ± 0.06
Mass (Kg)		68.8 ± 11.7	65.6 ± 8.9	65.4 ± 9.7	66.8 ± 10.1
Dual X-Ray absorptiometry derived data	Sarcopenic index	6.32 ± 0.81	5.97 ± 0.80	5.92 ± 0.81	6.08 ± 0.80
Sarcopenic Categorization	Proportion classified as Non-sarcopenic (Pre-sarcopenic/Low functional performance)	85% (0%/15%)	77% 23%/0%)	88% 12%/0%)	83% (12%/5%)
Physical Behavior classification	Proportion classified as Sedentary (Non-sedentary)	92% (8%)	100% (0%	75% (25%)	91% (9%)
Physical Behavior	Sedentary Behavior (h/24h)	9.6 ± 1.3	9.5 ± 1.0	8.3 ± 1.8	9.3 ± 1.4
	Light intensity physical activity (h/24h)	2.0 ± 0.8	2.1 ± 0.4	2.2 ± 0.7	2.1 ± 0.5
	Moderate to vigrous physical activity (h/24h)	3.0 ± 1.0	2.5 ± 0.8	3.6 ± 1.1	3.0 ± 1.0
Participant characteristics values are means ± SD.

Table 2. Changes in skeletal muscle-tendon size, muscle architecture, lean body mass, and functional performance.

		SBF (n=13)			LIPA (n=13)			Control (n=8)
		Pre	Post	Absolute change (Δ% change)	Pre	Post	Absolute change (Δ% change)	Pre	Post	Absolute change (Δ% change)
Gastrocnemius Medialis	Total Volume (cm3)	195.1 ± 33.3	186.2 ± 41.6	-8.9 ± 27 (-5 ± 13%)×	215.0 ± 49.0	211.2 ± 46.0	-3.8 ± 21.2 (-1 ± 9%)×	175.0 ± 31.6	196.3 ± 30.5	21.3 ± 12.6 (13 ± 8%)×
	FPA (°)	20 ± 3	19 ± 2	-1 ± 4 (-2 ± 22%)	18 ± 3	18 ± 2	0 ± 2 (-1 ± 9%)	18 ± 3	19 ± 3	1 ± 3 (0 ± 16%)
	Fascicle Length (cm)	5.5 ± 0.7	5.2 ± 0.6	-0.2 ± 0.6 (-4 ± 10%)×	5.7 ± 0.7	5.9 ± 0.5	0.3 ± 0.4 (5 ± 8%)×	6.0 ± 0.7	6.0 ± 0.7	-0.1 ± 0.4 (-1 ± 7%)×
	PCSA (cm2)	36.0 ± 6.7	35.7 ± 8.2	-0.2 ± 6.3 (0 ± 17%)	37.5 ± 8.3	35.5 ± 8.0	-2.0 ± 4.5 (-5 ± 12%)	29.7 ± 5.8	33.4 ± 4.7	4.0 ± 2.5 (15 ± 12%)
Gastrocnemius Lateralis	Total Volume (cm3)	130.8 ± 30.7	134.9 ± 39.1	4.2 ± 25.3 (4 ± 19%)	135.2 ± 29.5	137.5 ± 25.4	2.4 ± 26.2 (4 ± 21%)	133.5 ± 28.6	138.3 ± 21.0	4.8 ± 22.0 (5 ± 13%)
Vatus Lateralis	Cross sectional area at 50% muscle length (cm2)	41.3 ± 9.1	41.7 ± 9.7	0.4 ± 5.3 (1 ± 14%)	37.8 ± 6.0	37.8 ± 10.5	-0.1 ± 7.2 (-1 ± 18%)	34.8 ± 8.2	33.4 ± 7.4	-1.4 ± 2.8 (-4 ± 9%)
	Total Volume (cm3)	464.2 ±191.55	446.6 ± 201.8	0.5 ± 88.9 (0 ± 18%)	448.1 ± 95.2	435.6 ± 128.8	-17.4± 100.0 (-4± 23%)	395.2 ± 79.21	402.2 ± 78.45	2.7 ± 31.0 (1 ± 7%)
	FPA (°)	19 ± 3	18 ± 3*	2 ± 3 (11 ± 21 %)	16 ± 2	16 ± 3*	-1 ± 3 (-4 ± 18 %)	14 ± 3	14 ± 2*	-3 ± 2 (-18 ± 9 %)
	Fascicle Length (cm)	5.4 ± 1.3	5.6 ± 1.3	-0.1 ± 0.9 (0 ± 16 %)	5.2 ± 1.1	5.4 ± 1.4	0.2 ± 0.5 (5 ± 9%)	6.3 ± 1.2	6.3 ± 1.8	-0.4 ± 1.0 (-8 ± 12%)
	PCSA (cm2)	89.8 ± 21.9	92.4 ± 24.1	4.2 ± 24.7 (5 ± 30%)	79.2 ± 16.1	73.6 ± 22.4	-7.9 ± 20.3 (-11±23 %)	58.3 ± 16.5	58.8 ± 30.5	3.8 ± 11.3 (4 ± 22 %)
Achilles Tendon	Average cross sectional area (cm2)	0.78 ± 0.19	0.80 ± 0.20	0.03 ± 0.10 (3 ± 13%)	0.74 ± 0.14	0.74 ± 0.17	0.03 ± 0.11 (4 ± 15%)	0.70 ± 0.17	0.67 ± 0.19	0.01 ± 0.07 (1 ± 9%)
	Cross sectional area at 0cm (cm2)	0.86±0.34	0.90±0.31	0.03±0.19 (11±40%)	0.77±0.22	0.79±0.18	0.02±0.31 (13±48%)	0.74±0.20	0.69±0.08	-0.05±0.26 (-7±45%)
	Cross sectional area at 1cm (cm2)	0.78±0.19	0.81±0.33	0.03±0.17 (4±24%)	0.82±0.19	0.82±0.25	0.02±0.11 (3±19%)	0.74±0.28	0.78±0.23	0.04±0.17 (3±24%)
	Cross sectional area at 2cm (cm2)	0.76±0.14	0.78±0.15	0.02±0.12 (4±17%)	0.81±0.16	0.79±0.29	-0.02±0.11 (-2±15%)	0.70±0.36	0.67±0.25	-0.02±0.15 (-1±21%)
	Cross sectional area at 3cm (cm2)	0.72±0.22	0.71±0.11	-0.01±0.12 (-1±19%)	0.65±0.18	0.65±0.21	0.01±0.07 (1±9%)	0.59±0.24	0.65±0.25	0.03±0.15 (3±26%)
Dual X-Ray absorptiometry derived data	Arms Lean Tissue (Kg)	1.86 ± 0.33	1.85 ± 0.29	-0.01 ± 0.15 (0 ± 8%)	1.80 ± 0.22	1.76 ± 0.24	-0.03 ± 0.08 (-2 ± 5%)	1.71 ± 0.24	1.76 ± 0.24	0.05 ± 0.07 (3 ± 4%)
	Legs Lean Tissue (Kg)	6.17 ± 1.24	6.06 ± 0.99	-0.11 ± 0.45 (-1 ± 6%)	5.93 ± 0.77	5.85 ± 0.71	-0.07 ± 0.20 (-1 ± 3%)	5.66 ± 0.83	5.66 ± 0.83	0.01 ± 0.19 (0 ± 3%)
	Total Lean Tissue (Kg)	39.14 ± 5.92	38.91 ± 5.59	-0.23 ± 1.46 (-1 ± 3%)	37.10 ± 4.07	36.70 ± 3.88	-0.0 ± 1.13 (-1 ± 3%)	36.75± 4.05	37.44 ± 4.00	0.68 ± 0.79 (2 ± 2%)
Functional Performance Measures	Gait Speed (m/s)	1.12 ± 0.29	1.16 ± 0.37*	0.06 ± 0.08 (6 ± 10%)	1.12 ± 0.13	1.21 ± 0.18*	0.06 ± 0.07 (6 ± 6%)	1.30 ± 0.10	1.29 ± 0.24*	-0.02 ± 0.12 (-2 ± 9%)
	Eyes open balance (s)	19 ± 11	20 ± 12	1 ± 6 (11 ± 46%)	27 ± 5	27 ± 5	0 ± 4 (6 ± 30%)	26 ± 5	25 ± 8	-1 ± 4 (-7 ± 20%)
	Eyes closed balance (s)	3 ± 2	3 ± 3	0 ± 3 (29 ± 85%)	4 ± 3	5 ± 5	1 ± 2 (33 ± 63%)	3 ± 2	3 ± 1	0 ± 2 (2 ± 56%)
Participant Characteristics values are means ± SD.
Boldened baseline values represent significant baseline differences. * represents a significant time effect. × represents a significant group×time interaction effect.

Physical behavior

All groups were significantly matched for sedentary behavior, LIPA, and MVPA at baseline (p≥0.05). There was no group×time interaction for sedentary behavior (p=0.41). However, a trend for an effect over time was observed (p=0.08, ɳ_p²=0.21) driven primarily by a decrease in both experimental groups (SBF: -2±15%, LIPA: -4±14%) in contrast to control (4±30%). Promisingly, 8 participants (24%) positively shifted classification from sedentary to non-sedentary (SBF: n=3, LIPA: n=3, CON: n=2) in response to the intervention, with the other 26 participants (74%) remaining stable in their category over time. Furthermore no significant effects were observed for MVPA (p≥0.05) (Please see table 1).

Sarcopenia categories

All groups were significantly matched at baseline for categories of sarcopenia status (p=0.18), where 82%, 12%, and 6% were categorized as non-sarcopenic, pre-sarcopenic, and low functional performance respectively (Please see table 1). Only one participant positively shifted sarcopenia classification from low-functional performance to non-sarcopenic in response to the SBF intervention, with all other participants remaining stable in their category over time.

GM, GL, VL volume and PCSA intervention-induced changes

GM volume showed no effect of time (p=0.47), no effect of group (p=0.22) but a group×time interaction (p=0.014, β=0.77, ɳ_p²=0.24), with an increase in the control group GM volume being the driver for this interaction (Figure 1A). The associated changes for each group are illustrated in Figure 1B–1D. Similarly, a group×time interaction trend was observed for GM PCSA (p=0.06, β=0.56, ɳ_p²=0.18), with the control group increasing on average (15±12%) in contrast to both SBF (0±17%), and LIPA (-5±12%). However, there were no time, group, or group×time interactions observed for CSA, volume, or PCSA, in the GL and VL (Please see Table 2 and Figure 2).

Figure 1. Changes in GM muscle volume from baseline to post-intervention. Panel (A) Group-dependent GM muscle volume (Mean ± SD) at pre (week 0) and post intervention (week 8). There was a significant group×time interaction (p = 0.014) for GM volume. Panels (B–D) represent individual participants changes from baseline to post-intervention, for the SBF, LIPA, and control groups, respectively.

Figure 2. Changes in VL muscle volume from baseline to post-intervention. Panels (A) Group-dependent VL muscle volume (Mean ± SD) at pre (week 0) and post intervention (week 8). Panels (B–D) individual participant changes from baseline to post-intervention for the SBF, LIPA, and control groups respectively.

GM and VL resting muscle architecture

GM fascicle length (Lf) exhibited a significant group×time interaction effect (p=0.04). The primary driver for this effect was the significant difference between SBF, in which Lf decreased (-0.16±0.55cm, -4±10%), and LIPA, in which it increased (0.35±0.40cm, 5±8%) (p=0.04). Furthermore, once corrected for baseline differences, VL FPA exhibited a significant time effect (p=0.010, β=0.75, ɳ_p²=0.20), but not a group×time interaction (p=0.20) (Please see table 2).

Achilles Tendon dimensions intervention-induced changes

Interestingly for the analysis of the 4 discrete tendon CSA sites, a single trend was observed toward a significant main effect for time at 1cm of AT length (p=0.08, ɳ_p²=0.22), but no group×time interaction effect (p=0.99), with all groups increasing to a similar extent (SBF: 4±24%, LIPA: 3±19%, Control: 3±24%). Similarly, average tendon CSA (average of 4 discrete sites) showed no time, group, or group×time interaction (Please see table 2).

DEXA derived body composition intervention-induced changes

None of the DEXA-derived outcome variables (total lean tissue, arms, legs, and sarcopenic index) exhibited main effects of group, time, nor group×time interactions.

Functional performance measures intervention-induced changes

Gait speed exhibited a significant main effect for time (p=0.003, ɳ_p²=0.36). Despite no significant group×time interaction effect (p=0.24), both SBF (0.06 ± 0.08m/s, 6±10%) (Please see Figure 3, Panel A), and LIPA (0.06 ± 0.07m/s, 6±6%) increased from pre to post (Please see Figure 3, Panel B), in contrast to control (-0.02 ± 0.12m/s, -2±9%) (Please see Figure 3, Panel C). However, no significant main effects were observed for postural balance ability.

Figure 3. Individual participants’ changes in gait speed from baseline to post-intervention. Panels (A–C) represent individual changes for the SBF, LIPA, and control groups respectively. Panels (D–F) represent the associations between the relative changes in VL Volume (X axis), and the relative changes in gait speed (Y axis) for the SBF, LIPA, and control groups respectively.

Associations between relative changes in muscle-tendon complex size and relative changes in gait speed

There was a significant positive association between % change in VL volume and % change in gait speed (p=0.006). Specifically, within the pooled analysis of all participants, the percent change from baseline in VL volume significantly (R²=0.18, p=0.006), accounted for 18% of the explained variance in relative change from baseline in gait speed. Following sub-analysis by group, the explained variance in gait speed significantly (R²=0.31, p=0.027) rose to 31% in the SBF group (Figure 3, Panel D), 45% in the LIPA group (R²=0.45, p=0.0006) (Figure 3, Panel E) with no significant variance in the control group (Figure 3, Panel F). Furthermore, there was a significant positive association between the % change in GM Lf and % change in gait speed (R²=0.24, p=0.004), accounting for 24% of the explained variance. Interestingly, when sub-analyzed by group such an association only persisted for LIPA (R²=0.50, p=0.009) and Control (R²=0.64, p=0.014), with both groups accounting for similar amounts of the explained variance (LIPA: 50%, CON: 64%). Finally, a significant negative association was observed between the % change in GM PCSA and % change in gait speed (R²=0.33, p=0.001), accounting for 33% of the explained variance. Following sub-analysis by group, the explained variance in gait speed only persisted for SBF (R²=0.46, p=0.010), and LIPA (R²=0.37, p=0.014). Both experimental groups accounted for similar amounts of the explained variance (SBF: 46%, LIPA: 37%). No other significant correlations were observed between relative changes from baseline in muscle-tendon complex or DEXA outcomes and relative changes from baseline in functional performance measures.

Materials and Methods

Thirty-four community dwelling elderly women voluntarily participated in the study (See table 1). Intervention studies manipulating sedentary behavior in older adults are few, and to the authors’ knowledge no published interventions have examined changes in muscle-tendon complex size or lean body mass. Therefore, estimation of required sample size to detect significant changes in the desired outcomes was based upon two points: (a) previous sedentary behavior interventions in older adults that have observed improvements to physical function, have utilized total sample sizes of ~25-38 [50, 52, 53] (b) previous low-intensity resistance training studies in older adults, deemed total sample sizes of 17 [35], and 18 [63], adequate to detect changes in tendon and muscle size respectively. The current achieved sample size of 34 older females, falls within this range. Participants were all recruited from the local community. The study was approved by the local university ethics committee [approval code: 230118-ESS-DG-(2)], and written informed consent obtained prior to any procedures being performed, in line with the declaration of Helsinki. Exclusion criteria included history of lower limb muscle/ tendon/ joint disorders in the past six months, or current suffering from any chronic health condition which could affect the participants ability to independently perform an intervention of increased activity (e.g. cardiovascular disease, uncontrolled diabetes, active cancer, current diagnosis of stroke, Parkinson’s disease, etc). Furthermore, participants who partook in structured progressive resistance training (free weights etc) were also excluded at baseline. Nevertheless, inclusion criteria comprised all habitual physical activity profiles (regardless of meeting recommended MVPA levels). Participants initially visited the laboratories to complete screening/ questionnaires, as-well as undergo familiarization to the gait speed and balance (functional performance) assessments. After seven days, participants returned to the laboratories and underwent overnight rested and fasted, ultrasonographic assessment of the (GM), Gastrocnemius Lateralis (GL), Vastus Lateralis (VL) and Achilles Tendon (AT). Segmental analysis of body composition using DEXA imaging, and functional performance assessments were also conducted. Participants were randomly allocated in a single blind fashion to one of three groups: 1) Sedentary behavior fragmentation (SBF) (n = 13), 2) Single bout continuous light activity (LIPA) (n = 13), or 3) Control i.e. no lifestyle change (n = 8). All measures were taken at weeks 0 and 8.

Dual X-ray absorptiometry scan

Participants arrived at the laboratory in a fasted state (10-h to 12-h overnight) and were taken into a private scanning room. Participants changed into a hospital style gown and had their height (to the nearest 0.01m) and body mass (to the nearest 0.1kg) measured using a stadiometer (Seca model 213 stadiometer, Seca, Germany), and digital scales (Seca model 873, Seca, Germany) respectively. A dual energy x-ray absorptiometry (DEXA) scanner (Hologic Discovery: Vertec Scientific Ltd, UK) was used to ascertain lean body mass. Briefly, participants were asked to lie in a supine position, avoiding any contact between the trunk and the appendicular mass [84] (whole body procedure, EF 8.4 lSv). The slow moving ‘arm’ of the DEXA scanner passed over the body over the course of 7 minutes. Hologic software was then used to draw segmental analysis lines through the skeleton along regions of interest (Arms, Legs, Total) [85, 86].

Ultrasonography

Participants lay in a prone position, and rested for ~20 minutes to avoid fluid shifts [87, 88]. The ankle joint was then secured in neutral angle (0°) against a footplate. Participants were asked to remain still and relaxed, as Brightness-mode ultrasound (MyLab Twice, Esaote Biomedica, Genoa, Italy) was performed. Discrete muscle sites were marked by drawing a line from the medial to the lateral border of the GM and GL, at 25, 50, and 75% of each muscle’s respective length. Proximal and Distal endpoints of the AT were also marked, and length markers drawn in 1cm proximal increments from the calcaneal tuberosity. A novel panoramic imaging technique (panoramic view) granted an image of the GM/GL heads and thus anatomical CSA. Ultrasound panoramic imaging has previously been established as a reliable and valid method of CSA assessment when compared against magnetic resonance imaging [89, 90], and is sensitive to detect hypertrophic and atrophic alterations [83]. Briefly, the probe was moved with a constant speed and light pressure across the leg, to avoid compression during scanning. A Velcro strap was loosely attached (again to avoid compression) around the lower leg at each length marker to ensure the probe maintained the appropriate path and angle during each scan. Lastly, all ultrasound acquisition parameters were monitored, and consistency reproduced between scans. The ultrasound probe (7.5MHz linear array probe, 38 mm wide), was held perpendicular to the muscle. Once processing, the ultrasound probe was moved along the marked pathway, from the lateral to the medial border of the muscle (for representative images, please see Figure 4A, 4B) This procedure was repeated three times at each muscle site. The ultrasound probe was then positioned along the mid-sagittal line, at 50% of the GM muscle length, in order to record resting muscle architecture. Images of both resting fascicle pennation angle (FPA) and resting fascicle length (Lf), were then analysed using ImageJ (1.45s; National Institutes of Health). Three fascicles (defined from the deep to the superficial aponeurosis) of the GM were recorded and the mean value of both FPA and Lf determined. Linear extrapolation of fascicles was carried out where fascicles extended beyond the reach of the probe, as described previously [84]. This method has previously demonstrated good validity and reliability [33, 91].

Figure 4. Representative ultrasound images following panoramic ultrasound imaging. Panel (A) represents a transverse image of GM CSA (outlined for effect) at 50% of muscle length, Panel (B) represents a transverse image of GL CSA (outlined for effect) at 50% of muscle length, and Panel (C) represents a transverse image of VL CSA (outlined for effect) also at 50% of muscle length.

AT length was measured as the distance from the distal gastrocnemius myotendinous junction to the calcaneal insertion. Subsequently, AT CSA was obtained from representative transverse images (Depth: 30mm; Frequency: 27Hz; Focal Points: 1) at 0, 1, 2 and 3cm, of AT length. Offline ultrasound analysis was performed using IMAGEJ (1.45 s; National Institutes of Health, Bethesda, MD, USA) in a non-blind fashion. Determination of tendon CSA using this method has previously demonstrated good validity and reliability [92, 93]. Participants then switched to a supine position, with the knee fully extended and the hip angle raised to 45°, on top of a 30cm platform. The proximal and distal insertions of the VL were identified and 50% of VL length marked on the skin. Three more panoramic images of the VL head and thus VL CSA were then obtained as described previously (for representative image, please see figure 4C).. VL muscle architecture (FPA and Lf) was then determined, as previously described for the GM.

Ultrasound reliability

The same sonographer performed all scans and demonstrated excellent intra and inter day reliability. Specifically for the panoramic CSA imaging of the GM, GL, and VL the Intraclass Correlation Coefficient (ICC) was ~0.98, and the Coefficient of variation (CV%) ~4%, when reliability assessments were carried out on a subset of participants (n=8, 24% of total sample), comparing familiarization values to pre-test. Good inter day reliability was also observed for VL muscle architecture, specifically Lf (ICC = 0.96, CV% = 5%), and FPA (ICC = 0.87, CV% = 5%). For the Achilles Tendon, good inter day reliability was observed when CSA was examined at 0cm (ICC = 0.87, CV% = 7%), 1cm (ICC = 0.93, CV% = 6%), 2cm (ICC = 0.92, CV% = 6%), and 3cm (ICC = 0.76, CV% = 8%), and average of all sites (ICC = 0.97, CV% = 3%), when comparing tendon CSA at familiarization with pre-test values. Finally, good inter day reliability was also observed for GM muscle architecture, specifically FPA (ICC = 0.80, CV% = 4%), and Lf (ICC = 0.91, CV% = 67%), in a sub sample of participants (n=7, 21% of total sample).

Calculation of muscle volume and physiological cross-sectional area

GM and GL muscles volumes were calculated by treating the muscles as a series of truncated cones [94, 95], through the construction of several CSAs taken at discrete muscle sites (25, 50 and 75% of GM and GL length). Each of the four truncated cones was calculated using the following equation:

$musclevolume=\frac{1}{3}\cdot d\cdot [a+\sqrt{(a.b)}+b]$

Where:

d is the distance between the two CSA’s (a and b)

The sum of the four cones provided muscle volume for GM and GL. VL muscle volume was calculated from a single CSA re-construction at 50% of VL length and extrapolated to calculate overall muscle volume. This method of calculating muscle volume from a single CSA has been validated previously [96]. Physiological cross-sectional area (PCSA) was then calculated for both GM and VL using the following equation, as described previously [84].

$PCSA=musclevolume\text{\hspace{0.17em}/\hspace{0.17em}}Lf$

Postural balance assessment

The single balance postural test is well established in research using older persons, with documented reliability [55, 97]. Participants performed a single leg balance test with their eyes either open or with visual feedback removed through utilizing blacked out goggles to isolate proprioceptive feedback [72]. Each participant’s postural balance was tested on the leg they self-perceived to be their strongest. A number of measures were in place during assessment: (i) the researcher was present during all balance assessments; (ii) a soft chair was positioned behind the participants as a safety measure; (iii) participants hovered their hands above a height adjustable physiotherapy bed to begin the test before placing their hands by their side; (iv) in the event they felt they were going to lose their balance they would immediately place their hands back on the bed. This also marked the end of a particular trial along with raising the arms above head height or putting the non-balancing leg on the floor. Trial duration (up to a maximum of 30s) was recorded using a stopwatch. Three trials were performed with ~60s rest in-between. The average of the three trials for eyes open and eyes closed was then reported for each participant. Inter-day reliability was excellent for eyes open trials [Intraclass correlation co-efficient (ICC): 0.97%], and good for eyes closed trials (ICC: 0.75).

Gait speed assessment

Gait speed was assessed through the timed “Up and Go” test (TUG) [98, 99]. In an attempt to reduce TUG variability [100], the height of an adjustable stool was standardized to the length of each participants lower leg (distance in cm from the tibio-femoral junction, to the bottom of the footwear). The time taken between rising from and returning to the seated position was accurately monitored with a modified pressure sensor (Tekescan, South Boston, USA), and corresponding software. The sensor was attached to the surface of the chair in a manner that allowed accurate timing (0.01s) but did not impede the participants comfort whilst seated. Once instructed, participants rose from the chair, and walked at a maximum self-selected pace up to a box marked out on the floor with masking tape (approximately 6m away), before returning to the seated position. Total time was divided by the total course distance (12m) in order to calculate average gait speed [metres per second (m/s)]. The test was repeated 3 times with 60s rest in-between, and the average of three trials reported. Gait speed assessment exhibited excellent inter-day reliability (ICC: 0.91).

Comprehensive sarcopenia definition

DEXA derived appendicular lean body mass was divided by body height to provide a relative indicator of muscle quantity, termed sarcopenic index. Previously determined cut off points for both sarcopenic index and gait speed [74], were then used to classify participants into one of four categories, 1. Non-sarcopenic (sarcopenic index ≥5.5kg/m² and gait speed >0.8 m/s), 2. Pre-sarcopenic (sarcopenic index <5.5kg/m² and gait speed >0.8 m/s), 3. Low functional performance (sarcopenic index ≥5.5kg/m² and gait speed ≤0.8 m/s), and 4. Sarcopenic (sarcopenic index <5.5kg/m² and gait speed ≤0.8 m/s).

Physical behavior interventions

The purpose of the two intervention groups was to manipulate the method for displacing sedentary behavior time with added daily LIPA (45-50 mins). Both intervention groups were provided with a booklet, which contained simple LIPA suggestions compiled from the compendium of physical activities [101]. Participants were explicitly told to continue performing any pre-existing MVPA routines (e.g., exercise classes, etc). Throughout the 8-week intervention period all participants received fortnightly home visits from a member of the research team, to check on the progress of the intervention. Participants daily sedentary behavior, LIPA, and MVPA were assessed at baseline and the final intervention week, with a thigh mounted GENEActiv original triaxial accelerometer (GENEA, Activinsights Ltd, Kimbolton, UK), and a previously validated algorithm [102]. Participants were classified as sedentary if average daily sedentary time was ≥8h/day, as sedentary time appears to be exponentially hazardous above this threshold [2, 3].

SBF group: Participants were told that the purpose of their intervention was to reduce the amount of time spent performing sedentary behavior (sitting, lying, or reclining) especially in prolonged uninterrupted bouts. Participants were instructed not to perform sedentary behavior for more than 30 minutes at a time, and that for every 30 minutes of sedentary behavior performed the participant should stand up and perform 2 minutes of upright LIPA (general ambulatory walking, side to side shuffling, washing dishes etc).

LIPA group: Participants were informed that the purpose of their intervention was to increase the amount of time spent performing LIPA whilst maintaining habitual routines. Participants were instructed to perform a continuous single bout of 45-50 minutes LIPA (general ambulatory walking, side to side shuffling, washing dishes etc), every day for the duration of the 8-week intervention.

Control group: Participants who were randomly allocated to the control group were specifically instructed to maintain their habitual routine. Control participants were told that the overall purpose of the study was to study the link between health and habitual activity profiles.

Statistical analyses

Statistical analyses were carried out using SPSS (Version 25, SPSS Inc., Chicago, IL, USA). Parametricity was checked through the Shapiro–Wilk test to determine data normal distribution and the Levene’s test to determine equality of variances between groups. If parametric assumptions were met, baseline group differences were examined by a one-factor analysis of variance (ANOVA) (SBF, LIPA, CON) with post-hoc pairwise comparisons conducted using the Least Significant Difference. The effects of the interventions were determined using 2×3 split plot ANOVA (2 phases and 3 groups) or 2×4×3 (2 phases, 4 anatomical sites and 3 groups) split plot ANOVA depending on the outcome variable. Furthermore, linear regression analysis was performed on the relative changes from baseline for each muscle-tendon complex/ lean body mass outcome, and the relative changes from baseline for each functional performance outcome. GM muscle architecture data was not collected for 2 participants, meaning such analyses were carried out on a sub-sample (n=32). In cases of heteroscedasticity in variances, the Greenhouse Geisser correction was applied. In cases of non-normal distribution within group comparisons were made using the Wilcoxon-Sign Rank test, whilst, between group differences utilized a Kruskal-Wallis non-parametric equivalent of ANOVA (SBF, LIPA, CON) with post-hoc pairwise comparisons examined by Mann-Whitney U test. Chi-squared analysis was used to investigate nominal variables. Data are reported as Mean±SD (or Median, IQR for non-parametric data). Statistical significance was accepted when P<0.05. Furthermore, a statistical trend was deemed to be present when P was in the range of between 0.05 to 0.10. Study power (β) and effect size (ɳ_p²) are also reported where P is significant.

Author Contributions

GOP, DT and KT designed the research; DG conducted the research; DG and GOP analyzed data; and DG and GOP wrote the paper. DG and GOP had primary responsibility for final content. All authors read and approved the final manuscript.

Acknowledgments

We would like to extend our gratitude to the Musculoskeletal Sciences and Sport Medicine Research Centre at Manchester Metropolitan University for funding this project and all our study participants for their support.

Conflicts of Interest

D Grant - no conflicts of interest. DJ Tomlinson - no conflicts of interest. P Kolic - no conflicts of interest. K Tsintzas - no conflicts of interest. GL Onambélé-Pearson – no conflicts of interest.

Funding

Financial Support was provided by the Vice Chancellor Scholarship at Manchester Metropolitan University.

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