# Effect of Muscle-Specific Fatigue on the Risk of Anterior Cruciate Ligament Injury in Females
## 2 Materials and Methods
### Experimental Procedure  
The experimental protocol utilized to investigate muscle-specific fatigue on ACL load is presented in Figure 1. A pre-post study design was selected to assess the effect of treatment on selected parameters in the following order: (1) baseline measurement (pre-DDM); (2) treatment: muscle-specific fatigue protocol (QF or HA fatigue), general warm-up (control condition: 20 min walking); (3) post-measurement, i.e., after fatigue treatment (post-DDM). Approximately 20 min was required to perform DDM after the fatigue treatment. Hence, we asked the participants to walk for 20-min to give a light intensity warm-up [36]. All participants underwent three experimental conditions and visited the laboratory three times. The order of the three treatments (QF or HA or control) was randomized, and the participants had at least a seven-day interval between each treatment.  
On the first day, the participants got informed regarding the task, goal, process, and potential risks involved in the experiment. Then, the participants went through a pre-structured warm-up routine for 30 min which involved whole-body stretching [37], walking at preferred speed, performing five sprint runs on a straight pathway, and DDM runs at a preferred speed for familiarization of the task. Then, standard laboratory attire and shoes were provided to the participants. A total of 51 reflective markers were attached to 19 joints and 27 tracking markers to different segments (for the detailed location of markers: Supplementary Table S1) [38; 39].  
* Baseline measurement: Once the participants were ready, they performed three trials of the pre-DDM task, which involved running at a speed of 4.5 \(\pm\) 0.2 m/s, 5 m behind the force platform, followed by a DDM on top of the force platform (Figure 2A). The trial was valid only when the subject's right foot geared at the front and landed on the force plate and with a left turn at a range of 35\({}^{\circ}\)-55\({}^{\circ}\) in the direction of travel [6; 18; 19; 40]. The running speed was controlled using 2 timing gates (Witty Microgate, Bolzano, Italy).
* Treatment: Muscle-specific fatigue treatment was performed using an isokinetic strength measurement system (Figure 2B) performing knee flexion/extension at a fixed angular velocity. Initially, the subject's reference value of the peak knee torque was established, taking the average peak torque value of 3 trials of knee flexion/extension at an angular velocity of 90 \({}^{\circ}\)/s. The reference value of the peak knee torque was determined before the pre-DDM task. The fatigue task of the QF muscle involved knee extension and flexion at a fixed angular velocity of 90 \({}^{\circ}\)/s and 300 \({}^{\circ}\)/s, respectively, whereas the angular velocity for extension (300 \({}^{\circ}\)/s) and flexion (90 \({}^{\circ}\)/s) was reversed for the fatigue task of the HA muscle [22; 41]. The onset of fatigue was set when the peak torque reached 50 \(\pm\) 2% three times consecutively, with a two-minute break between each trial [41; 42]. Participants performed walking for 20 min at their preferred walking speed to avoid any muscle injury during the post DDM trials for the control condition.  
* Post measurement: During the muscle-specific fatigue protocol, the post-DDM task was performed immediately once the knee peak torque drop was confirmed. For the control treatment condition, participants performed a post-DDM task immediately after completing the 20-min walk, using the same protocol as the pre-test DDM task. Only the first successful trial was used for further analysis considering the fatigue recovery period.