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.