Skeletal muscle plays a role in producing force for body movement and, therefore, the force that muscle can produce has frequently been used as a parameter to estimate skeletal muscle function. It has been recently argued that “reliable” skeletal muscle force can be evaluated in vivo but not in vitro because there are several issues in an in vitro experiment such as an excessive oxygen supply. A unique event called excitation-contraction (EC) coupling underlies skeletal muscle contraction, which is sequential process initiating membrane excitation, sequential Ca²⁺ release from sarcoplasmic reticulum (SR) and then myofibril contraction. To elucidate a mechanism underlying force changes in vivo, biochemically purified SR and myofibril had been used in most previous cases. However, such samples do not necessarily function in the same manner as that under physiological condition, because they lose an interaction with adjacent organelle. To shed light on the real mechanism underlying in vivo force changes, we have developed the technique combining in vivo force measurement and mechanically skinned fibers. In mechanically skinned fibers preserving normal EC coupling, each organelle function can be assessed separately. Here, we introduce our experimental system and the outcomes which our system has provided for the first time.