<h3>Research Objectives</h3> To clarify the relationship among myofiber shapes, myofiber cross-sectional area, and circularity parameter. <h3>Design</h3> Randomized controlled trial study. <h3>Setting</h3> Department of Physical Therapy, Japan University of Health Sciences. <h3>Participants</h3> 500 myofibers in soleus muscles of 11-week-old male Wistar rats. <h3>Interventions</h3> Soleus muscles of five 11-week-old male Wistar rats were collected as samples. Muscle samples were rapid-frozen in isopentane—cooled in dry ice and acetone—and sliced into 10-μm slices in a cryostat, then stained with hematoxylin–eosin. The NIH-ImageJ software was used to analyze the number of corners that were counted according to the proposed criteria and myofiber cross-sectional area (MFCSA) of 500 myofibers. <h3>Main Outcome Measures</h3> Relationship among the approximate shape of myofiber cross-section (MFCS), myofiber cross-sectional area, and circularity parameter. <h3>Results</h3> As for the approximate shape of MFCS, the proportion of pentagons was 41%, which was the highest among polygons. A weak positive correlation was noted between the corner count and MFCSA, which indicated that polygons with more number of corners were associated with larger MFCSA. <h3>Conclusions</h3> It was suggested that the MFCS of soleus muscle had many approximate shape of pentagons. Moreover, a correlation was observed between MFCS and MFCSA, suggesting that it was also associated with the relevant functional features. <h3>Author(s) Disclosures</h3> The authors declare no conflicts of interest associated with this manuscript.