The protein substructure of skeletal muscle fibers forms a diffraction grating with repeating units, termed 'sarcomeres'. A laser scanning system is described that maps the lengths of sarcomeres (SL) and the widths of the first-order diffraction lines (DLW) of permeabilized single fibers in real-time. The apparatus translates a laser beam (λ = 670 nm and w0 = ~75 μm) along the length of a fiber segment through 20 contiguous regions per sweep at 500 sweeps/s. The fiber segments (~1 mm long) were obtained from vastus lateralis muscles of humans by needle biopsy. During both passive stretches and maximum fixed-end activations, the mappings of SL and DLW of the fibers were extracted from the diffraction spectra. Heterogeneity of SLs was evaluated by computing the standard deviation ( σSL) of the 20 SLs measured during a single sweep. Compared with the σSL before a passive stretch, the increase of 5±0.5% in σSL after the passive stretch, indicated differences in passive length-tension relationships along the fiber. In contrast, no change, ~0.5±0.1%, was observed in DLW. Within 10s after the fiber was returned to its initial length, the shape of the SL profile returned close to pre-stretch conditions ( σSL = 1± 0.2%). Following maximum Ca2+ - activation of the fiber, the heterogeneity of the steady state SLs increased greatly (DLW up by ~300% and σSL up by ~100%). The scanning system provided high resolution tracking of sarcomere behavior single muscle fibers. Potential applications are for studies of the mechanisms of muscle fiber injury and injury propagation.