## 4 Discussion
Therefore, the major factor that affected the results of this study was the anisotropic physical properties of the skeletal muscle, as the skeletal muscle consists of a parallel arrangement of myofibrils, muscular fibres, collagen and elastic fibres, and fascicles that result in its anisotropic action [19; 21; 63; 64]. Furthermore, muscle stiffness measurements are sensitive to the angle between the axis of the transducer and the orientation of the muscular fibres. [PERSON]'s modulus is correlated with shear modulus only if the transducer is oriented parallel to the muscle fibres. The propagation of shear waves along the direction of the fibres is faster than the perpendicular one. In addition, the pennation angle of the muscle is also changed once the muscle is in different contraction conditions. Therefore, the relationship between the orientation of the transducer and muscle fibre direction may have affected the results of this study, especially when measured during voluntary contraction [19; 73]. During voluntary contraction, the muscle will become stiffer. Propagation of the shear wave in more rigid tissue is too fast to be detected for some ultrasound systems. Consequently, the stiffness value at the greater contraction of the muscle, which is nearly the level of maximum voluntary contraction, cannot be evaluated [19]. Currently, for the measurement of active muscle stiffness using an ultrasound elastography approach, the level of muscle contraction has been analysed only at a sub-maximum voluntary contraction of about 40%-70%MVC [18; 36; 74]. On the one hand, the MyotonPRO measurement showed a significant difference (\(p<0.05\)) between the mean dynamic muscle stiffness of males and females, both when resting and at all levels of muscle contraction. These results also support previous studies that suggest MyotonPRO can be used to detect different conditions of muscle contraction, which are theorized to reflect varying conditions of stiffness during functional activities. MyotonPRO has demonstrated the ability to discriminate between different muscle contraction intensities [47; 51; 52]. In addition, in this research, there was a moderate positive correlation between dynamic muscle stiffness and [PERSON]'s modulus. Therefore, MyotonPRO can be used as an alternative measurement to assess the muscle stiffness of the FDS muscle at rest and during muscle contraction. The potential limitations of this study are addressed. Firstly, the resting and contracting states of the muscle were not monitored using electromyography; instead, they were determined by a hand grip dynamometer. Accurately investigating the resting state of the muscle and the occurrence of muscle fatigue during data-gathering processes was challenging. Therefore, EMG could be combined for further study to monitor muscle activity. Secondly, the subcutaneous fat thickness has not been measured before to measure muscle stiffness using MyotonPRO and SWE. In this respect, MyotonPRO is not suitable for measuring muscle stiffness once muscles are covered with subcutaneous fat of more than 20 mm. Therefore, further studies should measure subcutaneous fat to control this covariate. Thirdly, a small sample size was used in this study, and the effect of age was not considered. A relatively wide range of ages of participants, varying from 18 to 60 years, could potentially encompass considerably different mechanical properties of the muscle; therefore, these could be considered for further study. Furthermore, this study focused only on healthy subjects, so further studies should include different types of subjects, such as athletes or people with neuromuscular problems, in order to compare and generalize the results for each population.