Force depression after active shortening has been observed in different muscle preparations. It has been assumed that force depression is caused by the development of sarcomere length nonuniformities after shortening. However, this hypothesis has never been investigated in a preparation where individual sarcomere lengths could be directly measured. Here, we investigated force depression in single myofibrils ( n = 11) and tracked simultaneously the changes in individual sarcomere lengths ( n = 60) before, during, and after shortening and after a purely isometric contraction performed at the final length. Shortening produced force depression in all myofibrils (mean ± SE; 30.9 ± 3.9%). During shortening, all sarcomeres shortened, but not by the same amount. Sarcomere lengths were nonuniform, with the same mean SD before (0.11 ± 0.06 μm) and after shortening (0.11 ± 0.06 μm) and after a purely isometric contraction at the final length (0.10 ± 0.05 μm). Furthermore, greater shortening magnitudes were found for sarcomeres that were long in the initial isometric configuration. Nonuniformities of half-sarcomere lengths were also the same before (SD = 0.13 μm) and after (SD = 0.14 μm) shortening. We conclude from these results that the development of sarcomere (or half-sarcomere) length nonuniformities does not play a major role in force depression. Rather, force depression seems an intrinsic property of individual (half-) sarcomeres and muscle contraction.