To measure the distribution of local contractile movements in the layer of the myotubes, the movement of the matrix pattern of micro-markers placed on the back surface of the thin film to which the myotubes were attached has been tracked in vitro. A matrix pattern of micro-markers (diameter 4 μm, height 2 μm, spacing 30 μm) was created on the back surface of a polydimethylsiloxane thin film (thickness 6 μm) using photolithography technology. Mouse myoblasts were seeded on the front surface of the thin film and incubated for 10 days to differentiate into myotubes. Periodic electrical pulses (amplitude 2 V, pulse period 0.5 s, pulse width 1 ms) were applied between the electrodes of the titanium wire immersed in the medium. The movements of both the myotubes and the markers were observed simultaneously with an optical microscope. Experimental results show that the distribution of movement for each marker is related to the local periodic movement of the myotubes layer. A thin film with matrix pattern micro-markers created on the back side may allow in vitro analysis of the distribution of local repetitive contractile movements in the myotubes layer.