Muscle force is typically proportional to muscle size, resulting in constant force normalized to muscle fiber cross-sectional area (specific force). Mice overexpressing insulin-like growth factor-1 (IGF-1) exhibit a proportional gain in muscle force and size, but not the myostatin-deficient mice. In an attempt to explore the role of the cytoplasmic volume supported by individual myonuclei [myonuclear domain (MND) size] on functional capacity of skeletal muscle, we have investigated specific force in relation to MND and the content of the molecular motor protein, myosin, at the single muscle fiber level from myostatin-knockout (Mstn−/−) and IGF-1-overexpressing (mIgf1+/+) mice. We hypothesize that the addition of extra myonuclei is a prerequisite for maintenance of specific force during muscle hypertrophy. A novel algorithm was used to measure individual MNDs in 3 dimensions along the length of single muscle fibers from the fast-twitch extensor digitorum longus and the slow-twitch soleus muscle. A significant effect of the size of individual MNDs in hypertrophic muscle fibers on both specific force and myosin content was observed. This effect was muscle cell type specific and suggested there is a critical volume individual myonuclei can support efficiently. The large MNDs found in fast muscles of Mstn−/− mice were correlated with the decrement in specific force and myosin content in Mstn−/− muscles. Thus, myostatin inhibition may not be able to maintain the appropriate MND for optimal function.—Qaisar, R., Renaud, G., Morine, K., Barton, ER, Sweeney, HL, Larsson, L. Is functional hypertrophy and specific force coupled with the addition of myonuclei at the single muscle fiber level?