2.3.2. Methods of Participant Preparation for Measurement Procedure

Each measurement session consisted of measuring ankle torque during plantar flexion and dorsiflexion using a custom-made electronic dynamometer. All measurements were performed in the same testing laboratory with a constant room temperature (22 ◦C), allowing for an acclimatisation period of one hour before measurements. All possible adverse effects and the complete measurement protocol [41] were fully detailed to the participants. Possible adverse reactions, such as muscle cramps/fatigue/pain, or any other physical/emotional discomfort, were written and verbally presented to the participants and followed by cessation of the session in case such reactions might appear during the measurements. The participants were encouraged to fully relax while maintaining a sitting position with their trunk resting on the chair back-rest. Knee and hip joints were kept flexed, and the examined foot was resting in a plantigrade position on the dynamometer plate. Using nonelastic fibre belts, the fixation of the foot and thigh were ensured. For accurate measurements, the thigh strap was fixed just above the knee joint using one rigid fixation belt, and the foot was fixed in place using a second rigid fixation belt placed just above the dorsal aspect of the metatarsal-phalangeal joints (MPJ). This specific fixation allowed for the foot and ankle to remain stable and the heel to remain fixed in place during all measurements, as shown in Figure 6a,b. The foot positioning considered the ankle joint axis of rotation (defined as the line passing through the ankle malleoli) being aligned with the dynamometer's pivotal point. The dynamometer's pivotal point is marked with a blue horizontal line, as represented in Figure 6b.

**Figure 6.** The participant's sitting position: (**a**) fixation of the lower limb using a rigid fixation belt over the thigh right above the knee joint and the knee joint angle settled between 90◦ and 110◦; (**b**) foot positioned on the dynamometer plate, with the ankle joint axis of rotation above the apparatus's pivotal line marked on the device pedal with a blue line, with fixation of the rigid belt on the dorsum of the foot just above the MPJs level.

A more flexed knee position [49] allowed for the better isolation of the foot's small muscles (flexor digitorum longus, flexor hallucis longus and the long toe flexor muscles) also involved in plantar flexing of the ankle. The strap fixation just above the MPJs level allowed for the attenuation of the anterior tibialis muscle impact as an ankle dorsiflexor.

After all of the participants understood the type of requested muscle efforts, a succession of contractions followed as an experimental trial for better participant acclimatisation. Voltage acquisitions started while the clinician vocally commanded the direction of the ankle movements. After the acclimatisation test, a trial of three consecutive MVICs were registered for each ankle while the participants actively plantarflexed and dorsiflexed the foot. The acquisition period for the three consecutive MVICs was settled for 32 s. To better prevent muscle fatigue, a two-minute recovery break was permitted between the measurements for all pedal inclinations and all muscle groups. We measured the passive moment at rest and ankle torque at 0◦, +5◦, −5◦ of pedal inclination during three consecutive MVIC of 5 s, each separated by 5 s relaxation time for both plantar and dorsiflexor muscle groups, resulting in 12 measurements for each participant. The selected pedal inclinations (0◦, +5◦, −5◦) corresponded to the same range of the participant's ankle joint. When the tibia's long axis was perpendicular to the ground, having the foot resting on the dynamometer at 0◦of pedal inclination, we defined the ankle as being in a neutral position (90◦ of ankle dorsiflexion). From the neutral ankle position, we considered 95◦ of ankle dorsiflexion when +5◦ of pedal inclination and 5◦ of ankle plantar flexion when −5◦ of pedal inclination.

All participants were measured during two separate six-hour sessions. One session comprised six hours of sedentary behaviour (further named static behaviour) while maintaining a prolonged sitting position. The other session comprised of six hours of active behaviour (further named active behaviour) defined by low and moderate levels of physical activity.

A two-week interval was allowed between the two separate sessions.

The static behaviour was represented by six hours of prolonged sitting posture, with only small breaks allowed for participant's urgent personal needs. The active behaviour consisted of six hours of mixed activities (short or long-distance walking, ascending/descending stairs, orthostatic postures) without exceeding the moderate level of physical activity.

Three ankle torque measurements were performed at three different moments during each individual session: at two, four and six hours. The first measurement on each session was performed after two hours of the initiation of static/active behaviour.

The ankle torque measurement recordings from both the first and second sessions were later used for data processing.

#### 2.3.3. Methods for Validation of Acquired Data

One validation procedure during the measurements and two validation procedures during the interpretation of the results were performed for all of the acquired data. Participant-related errors (due to indiscipline or improper clinician commands) appeared and were considered errors during the measurements. When such errors were encountered, new measurements were requested.

All recorded voltage time graphs resulting from the oscilloscope were inspected by the main researcher immediately after each performed measurement, and only valid measurements were selected. Errors were kept for statistical analysis without being considered valid measurements.

Participant errors derived from off-set instability, improper contraction/break time (s), muscle efforts not corresponding with the clinician's vocal commands, insufficient number of MVIC, fatigue, insufficiently sustained MVIC, pain or participant's errors derived from testator command are possibly seen during measurements, and some of the most commonly encountered errors are represented in Figure 7a–d.

By a simple analysis of the time graphs obtained with the developed application, the human or apparatus errors were easily recognised. Improperly recorded data derived from both participant and/or operator errors or apparatus errors were eliminated.

The obtained results from the three consecutive MVIC, the difference between the maximum obtained level of torque and the minimum obtained level of torque (defined as peak torque in Nm), were registered and statistically analysed.

#### *2.4. Other Clinical and Functional Performed Tests*

A hand grip strength (HGS) test [50,51] for both right and left hands was performed using a factory-calibrated Hand Grip Dynamometer KERN MAP 80 K1 [52]. The testation was in accordance with the manufacturer's instructions. The participants were asked to stand in an upright position having their testing arm resting close to their trunk and the elbow flexed at 90◦. Three consecutive maximal volitional hand grip contractions were requested using the dynamometer's 80 kg spring, and the maximal value of strength in kg was registered for the right and left hands. Right/left arm dominance was asked and registered.

Bilateral calf circumference (CC) in cm at the calf's greatest girth was registered after being measured using a flexible nonelastic ruler. To measure CC, the participants were asked to stand in an upright position having their feet apart at shoulder width with their body weight equally distributed on both legs [53].

The number of repetitions during the Calf Raise Senior Test (CRST) [54,55] was registered for all participants. The participants were asked, while barefoot with their knees in maximum extension, to raise their heels simultaneously as high as possible, maintaining the same range of movement for all repetitions. To ensure stability, keeping their fingers on a vertical wall was permitted. While CRST was performed, muscle fatigue and muscle pain appeared during testation, and the onset moment of both fatigue and pain were registered.

The Calf Raise Test (CRT) [56] was performed starting from a unilateral weight bearing stance, and the number of repetitions for both legs was registered. Participants were asked to unilaterally raise their heel as high as possible, maintaining the same range of movement for all repetitions. To ensure stability, keeping fingers on a vertical wall was permitted. Muscle pain and muscle fatigue that appeared during CRT and the onset moment of both fatigue and pain were registered.

The chair raise test [57] was performed, and the total number of repetitions of chair raises in one minute was registered. Participants were asked to repetitively fully stand up in an upright position having their legs apart with their body weight symmetrically distributed on both legs, and then to sit down on a chair and perform as many repetitions as possible.
