Introduction: Inter-sarcomere dynamics within muscle myofibrils underlie basic mechanisms of contraction. In this study, we used microfluidic perfusions to control the environment of sarcomeres within isolated myofibrils to directly investigate inter-sarcomere dynamics. Methods: Pre-calibrated micro-needles were used to hold single myofibrils from rabbit skeletal muscles. A micropipette was used to locally control individual sarcomeres or groups of sarcomeres. The experimental were recorded in real time and analyzed for measurements of force and changes in length in individual sarcomeres within a myofibril. Results:(A) We tested length dependence of inter-sarcomere dynamics. Three initial average sarcomere lengths (SLi) were tested: (I) between 2.4-2.65μm, (II) between 2.65-2.9μm, and (III) above 2.9μm. Activation of the target sarcomere induced the displacement of the adjacent sarcomeres and the micro-needles towards the activation point. Force produced by the myofibril, and the displacements of sarcomeres situated adjacent to local activation were larger at increasing SLi ((I) 1.01±0.03μm, (II) 1.17±0.03μm, (III) 1.28±0.04μm). (B) The displacement of adjacent sarcomeres was further increased when the myofibril was tested in rigor conditions (1.08±0.03μm) in comparison with relaxing conditions (0.92±0.04μm), suggesting that the interaction among sarcomeres is regulated by myofibril stiffness. (C) All sarcomeres comprising a myofibril produced similar active forces (21.07±0.56nN/μm2) in a given SLi. (D) Long myofibrils produce higher forces than short myofibrils, and also presented higher levels of non-uniformity among sarcomeres, affecting the final magnitude of myofibril shortening. (E) Sarcomeres contracting along the descending limb of the force-sarcomere length relation negatively affect the total myofibril shortening and force production. Conclusion: Inter-sarcomere dynamics requires the cooperative work of the contractile and elastic elements in the in order to transmit force from sarcomere to sarcomere.