BACKGROUND: Understanding the interplay of muscle activity in the upper face is crucial because it can significantly impact the effectiveness and safety of aesthetic treatments. Traditional injection algorithms typically focus on the general 2-dimensional and 3-dimensional anatomy of muscles, often neglecting the areas where muscles exert the greatest force during facial expressions. OBJECTIVES: The goal of this study was to analyze the location of greatest morphological change in the upper facial muscles including the procerus muscle (PM), corrugator supercilii muscle (CSM), orbicularis oculi muscle (OOM), and frontalis muscle (FM) during various facial expressions. METHODS: A total of 34 healthy young individuals (17 females, 17 males), with a mean age of 23.6 ± 2.4 years [range 20-30], were examined with magnetic resonance imaging (MRI) to assess the length, thickness, and width of the upper facial muscles (PM, CSM, OOM, and FM) for 5 different facial expressions: repose, anger, joy, surprise, and sadness. RESULTS: Facial muscle thickness is a key indicator of activity during expressions such as anger, joy, surprise, and sadness. During anger, the PM and CSM decreased in length and width but increased in thickness, whereas the FM passively contracted to stabilize the expression. The OOM showed increased thickness in its medial, inferior, lateral, and superior portions during various expressions, with specific regions activating differently depending on the expression, such as the medial and lateral parts during surprise and the inferior and lateral parts during joy. The medial third of the CSM was the most active region during contraction. CONCLUSIONS: Upper facial muscles-as either agonists or antagonists-act together during facial expressions to stabilize facial expressions, emphasizing the need to assess both groups in neuromodulator treatments. The medial third of the corrugator supercilii shows the most significant MRI changes, making it the primary target for injections.