## 2 Materials and Methods  
This case-control study was conducted to evaluate the changes of lower-limb muscles after a 6-min fast-walk protocol. Data were collected during the period from April 2017 to November 2017 in a motion analysis laboratory at National Yang Ming Chiao Tung University, Taipei, Taiwan. Figure 1 shows the study flowchart. Fifteen non-flatfoot participants and nineteen with flexible flatfoot, all between the ages of 20 and 65 years, were recruited. Individuals with flexible flatfoot were distinguished from non-flatfoot individuals, using a navicular drop test, which is commonly used in clinical settings, as it can reveal the ability of the MLA to act as a supportive structure. The definition of flatfoot in this study is greater than 1 cm height difference of the navicular tuberosity between subtolar neutral and relaxed stance positions [17]. The inclusion criteria for the flatfoot group were established as flatfoot by the navicular drop test. We excluded subjects who self-reported a history of lower-extremity injuries within the previous 6 months, any lower-extremity surgery, and occurrence of neurological disease prior to participation. Subjects with skin allergy related to alcohol swabs and electrode pads were also excluded. This study was approved by the ethics committee of National Yang-Ming University (YM105056E) and conducted according to the Declaration of Helsinki. All participants' written informed consent was obtained before the experiments. We recorded each participant's height, weight, age, sex, and dominant foot; only the non-dominant foot was tested in the experiment. International Physical Activity Questionnaire (IPAQs) were used to assess each participant's daily activity level. The 6-min fast walking on the treadmill was the fatigue protocol in this study. Participants were instructed to walk on the treadmill with self-selected speed for 3 min to familiarize and warm up with the task. To estimate the preferred walk-run transition speed, the treadmill speed was increased gradually. The preferred walk-run transition speed was reduced by 0.1 km/h as the test speed so that participants could maintain walking for 6 min during the protocol. A motion capture system (Vicon Motion Systems Ltd., Oxford, UK) with eight infrared cameras operating at 100 Hz was used to capture and determine gait cycle. The Plug-In-Gait model was used; however, the kinematic data would process for further study. A force plate (AMTI, Advanced Mechanical Technology Inc., Watertown, MA, USA) operating at 1000 Hz was used to synchronically record ground reaction forces during tasks. The heel strike of level walking was determined at the first frame at which the vertical ground reaction force was larger than 20 N. A sEMG system (MP150, BIOPAC Systems Inc., Goleta, CA, USA) operating at 1000 Hz was used to collect muscle activation data synchronically with the motion capture system. Before and after the 6 min of fast walking, the maximal voluntary isometric contraction (MVIC) in the tibialis anterior, gastrocnemius, abductor halluces, and peroneus longus would be measured. Two functional tasks, namely level walking and single-leg standing tests, were executed in the same order. Maximal muscle strength assessment was performed by using manual muscle testing with a handheld dynamometer. To test the tibialis anterior, the participant was supine on the treatment table, and the dominant leg with knee flexion and hip external rotation placing underneath the popliteal fossa of the test foot and test foot performed ankle dorsiflexion (Figure 2a). To test the gastrocnemius, the participant was asked to lie prone with knee flexion 90\({}^{\circ}\) and performed ankle plantarflexion (Figure 2b). Peroneus longus was tested in a side-lying position and the dominant leg with hip and knee in flexion position placing under the test leg. Then the participant performed foot eversion in this position (Figure 2c). For the abductor hallucis, however, the maximal muscle strength cannot be assessed by the handheld dynamometer.