Fascicle measurement methods do result in lengths that are significantly different

Fascicle length is a key anatomical variable used to estimate functional adaptations of muscles. Although a recent study concluded that the fascicles measured on different specimens using in situ measurement and chemical dissection yield results that are not significantly different, these two methods have not been systematically tested within individual specimens. To that end, we compare the two measurement techniques on contralateral sides of (1) carnivoran masticatory muscles and (2) rat leg muscles to evaluate their statistical comparability. Our results show that the two methods result in statistically significant fascicle length differences, with in situ measurements being significantly greater in architecturally simpler muscles across a weighted average of masticatory muscles and among individual muscles. This trend holds for the rat soleus muscles but is reversed for the architecturally more complex gastrocnemius, with chemical dissection resulting in greater fascicle lengths. This suggests that pennation may influence methodological compatibility, with more complex connective tissue potentially obscuring fascicle ends and architecturally simpler muscles being prone to stretching when measured in situ. Given the importance of this anatomical measure, these results have potentially wide implications. Although small muscles with complex connective tissue yield shorter fascicle lengths when measured in situ, in general, in situ measurements are significantly longer by a factor of 1.3. Comparing fascicles measured using these traditional methods and through imaging techniques like DiceCT and micro-MRI, which allow evaluation of intact specimens, may provide further insight into the true anatomical reliability of these methods that unambiguously produce significantly different measurements.