# Fatigue Effects on the Lower Leg Muscle Architecture Using Diffusion Tensor MRI
## 4 Discussion
An increase in the pennation angles of the most contributing muscles (plantarflexors) was expected but no significant changes were verified. These results showed that we did not entirely verify our first hypothesis, particularly regarding muscle volume and muscle cross-section areas variables. The tractography process enables the quantification of the length of the muscle fiber. Fiber length is a muscle parameter that is commonly estimated using ultrasonographic equipment [32, 43, 44, 45]. Ultrasonography and reconstruction methods have been widely used to determine not just fiber length but also pennation angles; however, they do not take into account the spatial variation and orientation of the muscle fibers. Ultrasonography is easier to use, faster and cheaper, but is limited to planar measurements and can only be used in superficial muscles. Moreover, when dealing with long muscle fibers, the length is not directly measured but is instead estimated via regression. Muscle fiber length can only be rigorously determined via the microdissection of individual fibers from the fixed tissues [46]. Additionally, the effects of exercise interventions on muscle fiber length measurements can vary between ultrasound assessments and extrapolation methods due to a lacking gold standard method for muscle fiber length measurement [47]. Another advantage of DTI is the ability to measure both fiber length and pennation angle through the mapping of the eigenvalues from the diffusion tensor, which gives us the direction of the fibers. The mapping of the diffusion tensor allows for the reconstruction of the muscle fibers (and subsequent estimation of the fiber length), assuming that there is a preferential direction for the water to diffuse across the muscle cells. We observed that the lengths of the fibers estimated through this process were higher than the ones reported in the literature regarding cadavers or live subject experiments. Despite the advantages of the DTI measurement, a gold standard for muscle fiber length measurements has not yet been defined, which might be a limitation of this study. After the exercise task, the length of the fibers significantly increased for the plantarflexors SOL, GM and GL. We believe that the increase in the water content that circulates inside the cells will facilitate water diffusion. Increasing the space between the microstructures may have increased diffusion in all the directions of the tissue as a consequence. This lower restriction of the diffusion in the longitudinal axis of the fibers may justify the increase in the \"apparent\" length of the fibers. Having this in mind, the \"apparent\" length of the fibers before the exercise may have been underestimated since the probability of the tracking being discontinued was much higher. Moreover, different stop criteria settings changed the resulting fiber length. We used the default curvature settings \(>20^{\circ}\). We must still interpret these results with caution, as no study of the reliability and sensitivity of the method was performed, which is one of the limitations of the study. Compared with the existing literature data from cadaveric studies [18], our results for the pennation angle showed higher values for TA (almost twice the pennation) and similar values for the other muscles. Compared with in vivo studies using ultrasonography, we found similar results for the anterior angle of pennation of the MG muscle, as well as for the posterior angle of pennation for the GL muscle and for both compartments analyzed for the posterior soleus muscle reported by [PERSON] and colleagues [48]. Regarding muscle length, our values were smaller for the four muscles (less than 1 cm) when compared with the same cadaveric database and also with the subjects assessed by [PERSON] et al. [15] using ultrasonography. However, our sample was a group of young females with different anthropometric characteristics and smaller segments.