Sarcomere length and first order diffraction line width were measured by laser diffraction during elongation of activated frog tibialis anterior muscle fiber bundles (i.e., eccentric contraction) at nominal fiber strains of 10%, 25% or 35% (n=18) for 10 successive contractions. Pilot experiments demonstrated that single muscle cells elongated by only 10% strain ruptured ~50% of the time and thus experiments were performed primarily on small fiber bundles. Peak muscle fiber stress during eccentric contraction for all three fiber bundle strains was initially 311.5±3.6 kN/m², did not vary significantly between strain groups (p>0.6) and decreased by ~20% over the 10 contraction treatment. In contrast, tetanic tension, measured just prior to each eccentric contraction, differed significantly among strain groups and changed dramatically during the 10 contraction treatment (p<0.01). Average maximum tetanic tension for the three groups measured prior to any treatment was 203.7±6.8 kN/m², but after the 10 eccentric contraction sequence decreased to 180.3±3.8 kN/m², 125.1±7.8 kN/m², and 78.3±5.1 kN/m², for the 10%, 25% and 35% strain groups respectively (p<0.0001). Addition of 10 mM caffeine to the bathing medium decreased the loss of tetanic tension in the 10% strain group but had only a minimal effect on either the 25% or 35% strain groups. Diffraction pattern line width, a measure of sarcomere length heterogeneity increased significantly with muscle activation and then continued to increase with successive stretches of the activated muscle. Line width increase after each stretch was significantly correlated with the lower yield tension of the successive contractile record. These data demonstrate a direct association and, perhaps, a causal relationship between sarcomere strain and fiber bundle injury. They also demonstrate that muscle injury is accompanied by a progressive increase in sarcomere length heterogeneity yielding lower yield tension as injury progresses.