Ultrasound-guided interfascial plane blocks started with the transversus abdominis plane block, and various types of block have been introduced sequentially. Although the human deep fascia structure is the target for this technique, the relationship between anatomy of deep fascia and spread of injectate has not been fully elucidated. We microscopically observed spread of injectate in multiple slides after ultrasound-guided transversus abdominis plane block using latex mixed green colored dye solution in a cadaver (image A). The targeted transverse abdominis plane containing nerves (N) and vessels (A, V) was fully stained by dye solution (image B). However, injectate also spread into internal oblique and transverse abdominis muscle layer via perimysium, which were continuous with epimysium of deep fascia (arrowheads, image C). Specifically, intramuscular spread into internal oblique muscle was more predominantly observed. Anatomically, deep fascia structure seems to be complex and varied. It is not a simple sandwich type multilayer structure and, more importantly, fascial plane is not a closed space. Therefore, spread of injectate is not limited within fascial plane; spreading into neighboring muscle layers cannot be perfectly avoided. Furthermore, morphological and densitometric differences and biomechanical change of related muscles and fascias make it difficult to predict the final extent of injectate spread. Most anesthesiologists performing interfascial plane blocks rely on spread of injectate within fascial plane represented by hydrolocation under direct visualization of ultrasound for successful block. However, the inconsistent nature of this technique should be considered, as hidden anatomical factors can potentially affect clinical outcome.