The accuracy of using serial ACSA scans from MRI for the measurement of muscle volume has been reported previously [19, 33]. The anatomical cross-sectional area (ACSA) was measured by an image processing software (Simpleware ScanIP, Synopsys Inc., Exeter, United Kingdom) throughout the excursion of each of the individual muscles composing the two muscle groups of interest: triceps surae and quadriceps. The distal and proximal insertions of each of the muscles analyzed were identified before starting the analysis. For each of the seven individual muscles of interest (quadriceps muscles (VL, VI, VM, RF) and triceps surae muscles (GL. GM, soleus)), segmentation was performed every eight slices (18.2 mm). In the first of this analysis, the ACSA of each muscle was tracked manually and measured digitally, and this was done for the three test sessions of a same participant by the same investigator. Furthermore, a grayscale thresholding of the MRI image was used to identify the aponeurotic limit. In a second step, visible fat and connective tissue were excluded from the measurement to only take into account the contractile part of the muscle volume [34]. To separate the non-contractile tissue, a region of interest (ROI) containing muscle and subcutaneous fat was made. Then, a histogram of signal intensity within the ROI was produced. To separate contractile and non-contractile tissues with minimal investigator bias, thresholding was performed by the Maximum Entropy method, a reliable histogram shape-based technique used in medical imaging analysis [35]. Pixel values above the threshold were considered to be non-contractile tissue [18]. Finally, this area, comprising fat and connective tissue, was then subtracted from the total muscle area, in order to measure only the CSA of the contractile components.

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