## 2 Materials and Methods
### Marker Protocol  
The present study was focused on the 3D kinematic analysis of the lower limb. The lower body was segmented as follows: one pelvis, two femurs, two tibias and two feet. A new marker protocol based on the PiG [24] protocol was developed for this study. The PP is a nonoptimal pose estimator based on a minimum number of markers placed on the subject. In total, 15 physical markers and 8 virtual markers were used (Figure 1). No interference was observed between the position of the markers of the PP and the bicycle in both the sagittal and frontal planes (Figure 2a,b). No kinematic constraints were imposed. The choice of physical marker locations was made with particular care to ensure that the markers were not hidden during pedalling. In addition, they were placed in areas with little movement of the soft masses. The pelvis kinematics were modelled by placing two markers on the posterosuperioriliac spines (RPSI and LPSI) and one marker on the sacrum (SACR). The sacrum is a fairly large bone; so, to ensure that, for all participants, the location was equivalent, this marker was placed just above the intergluteal line. This disposition allowed us to eliminate the markers placed on the anterior iliac crests that, in the pedalling posture, were partially hidden and very influenced by the STA. Figures 1 and 2 show that in the thighs and legs, the markers placed in the fatty zones [24] were replaced by markers placed in the greater trochanter and in the tibial tuberosity, respectively. This change significantly reduces the sensitivity of the results to incorrect placement of the markers [24]. The marker arrangement in these solids is as follows: Thighs presented one marker placed on the greater trochanter (RGTC and LGTC) and another on the lateral epicondyle (RKNE and LKNE). The legs featured one marker placed on the tibial tuberosity (RTIB and LTIB) and another on the lateral malleolus (RANK and LANK). Following the arrangement proposed by [PERSON] et al. [24], two markers were placed on the foot: one on the calcaneus (RHEE and LHEE) and one on the second phalanx of the first metatarsal (RTOE and LTOE). When the participants are wearing shoes, it can be difficult to locate the two anatomical points where the markers should be placed. However, a procedure was developed that only requires the detection of one of the anatomical points. Firstly, a static capture was taken of the participant's bare foot with the foot markers placed on the specified landmarks. Once the capture was made, the relative position between the foot markers was obtained. Depending on the participant, the HEE marker was sometimes easier to detect by feeling inside the shoe, while on other occasions, the TOE marker was easier. Using the computed relative position of the known marker, the location of the other foot marker could be obtained. Figure 1: Markers’ location that form the protocol. Green: physical markers. Red: virtual markers. Figure 2: Markers placed on a participant during pedalling. (**a**) Marker position during pedalling in frontal plane; (**b**) marker position during pedalling in sagittal plane. As previously mentioned, the protocol incorporates eight virtual markers. These markers are not physically placed on the participant during motion capture, which enhances participant comfort. They are added during the post-processing phase to define joint centres and axes of rotation. In the present study, virtual markers were employed to define the hip and ankle joint centres, as well as the knee axis of rotation. The hip centre (RHJC and LHJC) was calculated by the functional method SCoRE [29]. The knee rotation axis, which consisted of the knee joint centre (RKJC and LKJC) and an arbitrary point of the knee rotation axis (RKJA and LKJA), was determined from the functional methods SARA [30]. The ankle joint centre (RAJC and LAJC) was determined from the methodology developed by [PERSON] et al. [24]. A table explaining in detail the position of each marker in the PP was included in the Supplementary Materials.