## 2 Materials and Methods
### Arterial Stiffness  
The subjects were instructed to rest quietly in the supine position at least 10 min before measurement. Brachial blood pressure, brachial-ankle pulse wave velocity (baPWV), and carotid-femoral pulse wave velocity (cfPWV) were obtained using an automated vascular testing device (VP-1000 plus, Omron Healthcare). All measurements were made in duplicate, and average values were used for subsequent analyses.  
The carotid _uf_PWV measurements were performed on the right carotid artery by using a Prodigy ultrasound imaging system with a 6.4 MHz linear array probe (S-Sharp Corporation, New Taipei City, Taiwan). The depth was fixed at 70 mm. Pulse repetition frequency (PRF) was set at 1K Hz. The number of received beams was fixed at 128. After confirming the location of the arterial wall (common carotid artery 1-2 cm proximal to the right carotid bulb), 128 channels were used simultaneously to generate two cycles of plane waves at a speed of 1540 m/s. Before the operation, we first used B mode to confirm if the carotid arterial wall images were clear. Subsequently, the participants were requested to hold their breath for 2 s during exhalation to acquire data for offline analysis as previously  
Figure 1: Muscle stiffness measurement. (**A**) The ultrasound probe was placed on the mid-point of the biceps brachii of the upper arms; (**B**) the pixel tracking of two different lines (\(\sim\)1 cm apart) was analyzed by the software (Muscle Tracer, S-Sharp Corp. Taiwan); (**C**) the average strain rate and area under the curve (AUC) calculated from two lines throughout the protocol were analyzed.  
described [19,23]. We specifically instructed participants to avoid force exertion of the chest wall while breath-holding to eliminate possible intrathoracic pressure elevation and arterial pressure changes. The coefficient of variation for _uf_PWV measurement in our laboratory was below 5%.  
We measured the distance of the arterial wave by measuring the longitudinal velocity of the vessel wall to calculate the arterial velocity. A similar methodology was reported in the previous study [20]. Briefly, we used B-mode images for this measurement because the vessel wall signal is stronger than the blood signal, and therefore a more significant reflected signal can be seen on the ultrasound image (Figure 2A). In this study, the phase tracking method was used to demodulate the RF signal of each channel (128 channels in total) in the image after receiving delay and sum beamforming, and to obtain the complex signal (I-Q signal) in the fundamental frequency, which can be used to estimate the longitudinal velocity of the tube wall in the channel [24]. As shown in the left panel of Figure 2B, we connected the foot positions as the black line from 128 channels, followed by a robust bi-square linear regression analysis. The advantage of using a complex signal is that the phase change can be easily calculated and estimated. The systolic foot was defined as time points where the acceleration waveform reaches a maximum before and the systolic peak of the waveform. Carotid _ufPWV_ velocity at the end of diastole (_ufPWV_-dia) and systole (_ufPWV_-sys) were calculated as the reciprocals of linear regression slope, respectively (right panel of Figure 2B).  
Figure 2: (**A**) B-mode images of common carotid artery obtained by the ultrafast ultrasound machine; (**B**) the left panel illustrates artery diameter change obtained from each channel (filled circle = diastole; triangle = systole). The right panel represents the linear regression after receiving signals from 128 channels. _Red_ represents movements toward the probe. _Blue_ represents movements away from the probe. Color brightness represents speeds of movements, where the brightest red line indicates the fastest pulse wave (systole). The reciprocals of symbol line slopes represent _uf_PWV-dia (filled circle) and _uf_PWV-sys (triangle), respectively.