## 2 Materials and Methods
### Experimental Procedure  
The three target muscles of the lower extremity were Rectus Femoris (RF), Tibialis Anterior (TA) and Gastrocnemius Medialis (GM) of right side. All three muscles were tested under two positions: resting (RST) and mid-range lengthened (MRL). Under both positions, subjects were asked to maintain the still and relaxed state, to avoid any movements that would induce muscle contraction and consequently change the read-out values. Since no active muscle movement was involved, under both positions, the muscular architectural change was induced by changing the muscle tension from the resting state to the mid-range lengthened state (see Figure 2) for the probe positioning on the reference muscles under two testing positions).  
Figure 1: An example of typical elastography images of tibial anterior muscle at resting position. Q-BoxTM: the Aixplorer scanner software for measuring the shear wave modulus in the region of interest (white circle in the upper middle of the figure); SD: standard deviation.  
The positioning of the ultrasound probe was standardized and applied consistently throughout all experiments. The general procedure for the correct probe positioning were as follows: (i) find the body landmarks, and draw a line in between (Figure 3a); (ii) mark the point on the line which is corresponding to the target muscle, and draw a transverse line on it (Figure 3b); (iii) place the probe along the transverse line, and observe the target muscle with the conventional B-mode image; (iv) mark the medial and lateral border of the target muscle on the skin (Figure 3c); (v) change the probe orientation to align with muscle' contraction direction, while the probe remaining in the middle of the medial and the lateral border; and (vi) ask subject to contract the muscle, to verify that the position is positioned on the right muscle. These probe positioning steps were consistent for all three muscles, while the landmark points differed accordingly to the specific tested muscle.  
For RF muscle, the body landmarks were anterior superior iliac spine and the superior border of the patella, with the transverse line drawn in the middle of the line between landmarks [16]. The subject was lying in supine position on the examination table. For the RST position, the subject was in supine position, with the knee in the neutral position on the examination table (Figure 2a). For the MRL condition, the subject maintained supine position, with the knee off the table at 90 degrees flexion (Figure 2b).  
For TA muscle, the body landmarks were fibula head and lateral malleolus, with the transverse line drawn on the upper one-third of the line between landmarks [23]. The subject was lying in supine position on the examination table. For the RST condition, the foot was positioned against the wall at 90 degrees dorsiflexion (Figure 2c). For the MRL condition, the subject's ankle was off the table from the above malleoli, and the food dropped without extra force applied (Figure 2d).  
For GM muscle, the body landmarks were the popliteal fossa and the cross point of Achilles tendon and two malleoli, with the transverse line drawn on the upper one-third of the line between landmarks [24]. The subject was lying in prone position on the examination table. For the RST condition, the foot was positioned on the bed and maintained relaxed (Figure 2e). For MRL condition, the ankle was off the table, with the foot kept against the wall at 90 degrees (Figure 2f).  
Figure 2: Probe locations for each muscle and position. (**a**) Rectus femoris at resting (RST) condition; (**b**) rectus femoris at mid-range lengthened (MRL) condition; (**c**) tibialis anterior at RST condition; (**d**) tibialis anterior at MRL condition; (**e**) gastrocnemius medial at RST condition; (**f**) gastrocnemius medial at MRL condition.