The main goal of this study was to evaluate the dynamics of sarcomeres during isometric activation of skeletal muscle myofibrils. Rabbit psoas myofibrils (n=14) were attached between a pair of cantilevers for force measurements at one side and a rigid glass needle at the other side, and their images were used for measurements of individual sarcomere lengths (SL) during contractions. Myofibrils were set at average SL between 2.13 and 3.06 microm, and were activated and held isometric for 20-35s during which SL and force were continuously measured. SL dispersion increased from the rest state to activation, but it remained mostly constant during the activation period. Even with the length non-uniformity developed during myofibril activation, most sarcomeres stabilized their length changes during the isometric contraction. As a result, sarcomeres contracted at different degrees of filament overlap while producing similar forces. When the myofibrils were separated in two groups that produced force at averaged short (< or =2.5 microm) or long (> or =2.5 microm) SL, the initial non-uniformity was greater in long lengths, but changes observed in sarcomeres during the activation period were similar, suggesting that sarcomere stability is not length-dependent.