Participants

Published recommendations of sample size requirements for reliability studies vary, but 15–20 participants has been suggested as sufficient²⁵, and previous studies with Myoton have used 20 or fewer participants^26,27,28. Twenty healthy participants were recruited (n = 10 males, n = 10 females) from staff and students at the University of Southampton (age 28.95 ± 2.77 BMI 24.28 ± 1.47) took part in the study. Ethics approval was obtained from the Faculty of Environmental and Life Sciences Ethics Committee (no. 40307), University of Southampton. All methods were performed in accordance with the relevant guidelines and regulations. Posters were circulated throughout the faculty advertising the study. Informed consent was obtained from all subjects prior to testing. Participants were asked to attend two sessions, the first lasting approximately two hours, and the second a week later lasting one hour held in the afternoons²⁹. The room was set to an ambient temperature 22–24 °C, warm clothes worn during measurements, to avoid temp induced tonus changes.

Inclusion criteria

Over the age of 18 up to the age of 40; able to understand English.

Exclusion criteria

Musculoskeletal injury or surgery in the last five years which led to immobility for more than one week; uncontrolled diabetes or blood pressure; a known neurological disorder; arthritis restricting ability to perform everyday activities; receiving treatment for cancer; taking medication which affects muscle function. No scars on MP sites, no obvious inflammation/reddening of the skin, no underlying disease leading to spasms/paralysis (Parkinson’s disease, Huntington’s disease, stroke, nerve damage, etc.), no tonus reducing or enhancing agents were taken 12 h previous to the measurement (muscle relaxants, coffee more than 1 cup, alcohol, drugs, etc.), Participants were asked not to undertake any strenuous exercise within 12 h before the data collection session, including their mode of transport to the session (e.g. to take public transport or drive, as opposed to walking or cycling).

Measurement points

The MP for this study were based on those for the Myotones project currently ongoing on the ISS²⁴, where both technologies are tested at 11 sites (seven of which are assessed in this study). All MPs (all on right side) can be seen below (Fig. 1).

As some of the MPs listed below included in the Myotones study already have well established reliability, only seven measurement points were examined in the present study: MP1, MP2, MP3, MP6, MP7, MP9, and MP10.

Participant in prone lying: with roller under the ankle

1. 1.

   Plantar fascia (MP1)—palpate the fascia (tight band) from the middle arch of the foot up towards the heel until the feel of the band is lost. MP1 is 1 cm down from the base of the heel (confirmed with ultrasound)—ultrasound image taken longitudinally.

2. 2.

   Achilles tendon (MP2)—thinnest part of the tendon (palpatable using thumb and index finger as “forceps” or “tweezer grip”)—ultrasound image taken longitudinally.

3. 3.

   Soleus (MP3)—Measure the distance between MP2 and middle of the popliteal crease at the back of the knee. MP3 is located 33% up the back of the calf from MP2, then 3 cm medially—ultrasound image taken longitudinally.

4. 4.

   Gastrocnemius (MP4)—from MP2, measure 66% up the back of the calf, then 3 cm medially—ultrasound image taken longitudinally.

5. 5.

   Multifidus lumbar L4 (MP5)—draw the line (tape measure) between the top of hips (Iliac crests of pelvis) to find level on the backbone. From the mid-point on the spine, measure x = 1 cm to the right—ultrasound image taken transversely.

6. 6.

   Transversus abdominis (TrA; 5.1)—find the Umbilicus (belly button), move laterally until the three lateral abdominal muscles (external oblique, internal oblique, TrA) are seen as parallel (ultrasound only).- ultrasound image taken transversely.

Participant in sitting

1. 7.

   Splenius capitis (MP6)—tape measure between the base of the external occipital protuberance (bump at back of the skull base) and the Acromion (bump on top of shoulder) and mark the centre of the line. Tape measure between C7 and angle of the jaw (mandible) just below the ear and mark where the lines cross. From this intersection, move 3 cm forward from this cross (confirmed with ultrasound).—ultrasound image taken transversely.

Participant in supine lying: with roller under the knee

1. 8.

   Deltoid anterior (MP7)—place a tape measure from the Acromion down to the crease on front of elbow. Put your finger on the tape at the same height as the crease of the armpit and move your finger up 2 cm. To confirm the point, ask the participant to raise their arm, and the muscle should contract under your finger—ultrasound image taken transversely.

2. 9.

   Rectus femoris (MP8)—tape measure in a line formed between the superior border of the patella and the iliac spine (bump on front of right side of pelvis). From the patella, measure 33% up of the total distance between the above two points—ultrasound image taken transversely.

3. 10.

   Infrapatellar tendon (MP9)—Locate the inferior edge of the patella in the middle and the tibial tuberosity (bump on top of the tibia or shin bone). Measure 50% of distance between the two points—ultrasound image taken longitudinally.

4. 11.

   Anterior tibialis (MP10)—measure the distance from the lower edge of the patella to the middle of the ankle joint between the medial and lateral malleoli. Measure 50% of the distance between these two points. Then move 2 cm to the outside of the leg over the muscle belly (Tibialis Anterior)—ultrasound image taken transversely.

MyotonPRO device

A smartphone-sized, non-invasive digital palpation device (MyotonPRO) was used to assess biomechanical properties of muscles (Myoton AS, Estonia). The MyotonPRO device applies mechanical impulses to the skin (duration 15 ms, force 0.4 N) under a pre-compression force of (0.18 N) on the tissue layer of interest to minimize signal bias from soft tissue overlying muscle and tendon. The device is held perpendicular to the skin ± 5° (checked by MyotonPRO device). The impulses cause damped oscillations of the underlying tissues, which are recorded as parameters for tone (represented by frequency, Hz), stiffness (N/m), and elasticity (Logarithmic Decrement). There is a small mark on the probe to show how far to push down on the skin and once the pre-compression load is met, an indicator light changes from orange to green and the preset impulses are applied automatically. The MyotonPRO device records the coefficient of variation (CV) between the sets of at least five different mechanical impulses per measurement and displays this as a percentage next to each parameter. In the present study a threshold of 3% CV was set, if any parameters were over this threshold the measurement was repeated.

Ultrasound imaging

Images were taken using a real-time B-mode ultrasound scanner (ORCHEO lite, SONOSCANNER, Paris, France; designed by CNES, CSA and ESA, referred to as the ECHO device) with a 3.5–16.7 MHz linear transducer. The transducer was placed transversely or longitudinally on the skin, depending upon the MP being imaged (see specific sites above), with minimal pressure to avoid compression of the underlying tissues. Ultrasound images were taken in accordance with the directions stated above, with one image taken at each point.

All images were measured later off-line by one investigator (PM), using a Matlab algorithm (written by MW). Ultrasound imaging of musculoskeletal soft tissues is known to be reliable^30,31 and valid^32,33 against the gold standard of magnetic resonance imaging (MRI). As with the Myoton technique, standardization of factors influencing recording of images is important¹⁴.

Operator experience

The number of years of experience for the three researchers collecting data was: Researcher 1: Ultrasound Imaging 5 years, Myoton technology 5 years; Researcher 2: Ultrasound 13 years, Myoton technology 9 years; Researcher 3: Ultrasound over 30 years, Myoton technology 9 years.

Experimental procedure

During the first session one operator initially located the anatomical measurement points (MPs) and marked them on the participant lying on a gurney at full relaxation. The measurements were recorded in a logbook for each participant ahead of the next session. Three independent operators blind to the other recordings then undertook the MyotonPRO and ultrasound measurements. Raters were not required to re-mark the participants within the same sessions, so reliability of the data acquisition was evaluated and not the whole Myoton or ultrasound procedure.

For the MyotonPRO recordings, two sets of five impulses were applied as described above. Coefficient of variation measurements for each variable on the device were inspected and if any were higher than 3% the measurement was retaken. All points were recorded twice and a mean of the 10 pulses was taken and used in the analysis. Ultrasound images were taken in accordance with the directions stated above, with one image taken at each point.

For the between days intrarater reliability, participants were invited back a minimum of a week later for the second session (in the afternoons), where only measured by one operator repeated both ultrasound and MyotonPRO measurements, after relocating the same MPs as in the previous session.

Data analysis

Interrater reliability was assessed using a two-way mixed, single score intraclass correlation coefficient (ICC) (3,1)³⁴.

Intrarater reliability between days was assessed two-way mixed, single score intraclass correlation coefficient (ICC) (3,1)³⁴.

Intrarater reliability within sessions was assessed using a two-way mixed, average score intraclass correlation coefficient (ICC) (3,2) to compare between the two sets of five impulses.

All statistical tests were performed using SPSS (v 26, Armonk, NY: IBM Corp), with the alpha value set at 0.05.

The guidelines for interpretation of ICC results were taken from Koo and Li³⁰ with below 0.5 = poor, between 0.5–0.75: moderate, between 0.75 and 0.90 = good, and above 0.90 excellent.

Matlab programme for measuring scans

Ultrasound scans were measured with a custom written graphical user interface (GUI) created in Matlab (Mathworks, USA) using bespoke functions (MW). The GUI allowed the investigator to import the bitmap images obtained from the ultrasound scanner. The GUI calibrated the images by determining a scaling factor between the number of pixels and a 1 cm distance obtained from the scale displayed on the side of the ultrasound image. The GUI then allowed measurements to be made on the ultrasound image by the investigator through identifying and clicking on various landmarks. Specific landmarks and measurements are detailed below for each muscle and tendon. Distances were then calculated as the Euclidean distance between landmarks, then converting to centimetres using the scaling factor and then exported to an Excel file.

Scans were calibrated against the scale on the side of the ultrasound scan, marking a measurement of 1 cm with a cursor.

Passive muscle thickness was measured on cross-section planes of ultrasound scans between the muscle’s borders from the bottom of their superior fascia to the top of the inferior fascia.

Tendon/fascia thickness was measured by creating a 1 cm wide box, then creating a line on the top of the structure and a line at bottom. The distance between the two lines was then automatically measured 100 times and the mean thickness measurement was taken.

Subcutaneous tissue thickness was measured from the top of the skin to the superior border of the muscle fascia (that is regular peripheral body fascia structure with compartment fascia and epimysial layers considered as one structural layer (i.e., muscle fascia) of regional variance however all these structural layers are however below USI image resolution). A second measurement was taken from the superior border of the muscle fascia to its inferior border. The thickness of the fascia was included with fat thickness in the subcutaneous tissue measurement (Fig. 2). The deep foot extensors (MP10 deep foot extensors)- only measured with ultrasound using the same subcutaneous tissue thickness as MP10, but with the muscles measured as one complex all the way to the tibia.

Example of ultrasound, and measurement points for data analysis. (a) Plantar Fascia, (b) Achilles tendon, (c) Soleus, (d) Splenius capitis, (e) Anterior Deltoid, (f) Patellar tendon, (g) Anterior tibialis.

Minimal detectable change (MDC)

The MDC gives a meaningful and practical assessment of measurement error, providing a single value for each variable in the unit of relevant measure³⁵, which is the smallest change in score that occurs due to error and not likely related to chance variation in measurement³⁶. The MDC based on the SEM was calculated using the following formula outlined in Haley and Fragala-Pinkham³⁶: MDC = 1.96 (z scorelevel of confidence) × √2 × SEM). The formula to calculate SEM was SEM = SD × (√(1 − ICC)) as outlined in Wilken, Rodriguez³⁵.

Bland and Altman

To complement the ICC reliability data, the Bland and Altman test with limits of agreement was used and graphed (Supplementary Table 1)³⁶. This test was conducted between each researcher (R1–R2, R2–R3, R1–R3), and for the between day testing.