The purpose of the present study was to 1) develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers; and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers from Xenopus laevis were either electrically stimulated for 15 min of tetanic contractions (EX; n=26) or not stimulated to contract (REST; n=14). The relative mean developed tension of EX fibers decreased to 29±7% of initial peak tension at the stimulation end-point. Individual fibers were then allowed to recover for 1 hr (n=9), 2 hr (n=8) or 4 hr (n=9) prior to isolation of total cellular mRNA. HSP72, heat shock protein 60 (HSP60) and cardiac -actin mRNA content were then assessed in individual fibers using qPCR. Relative HSP72 mRNA content was significantly (p<0.05) elevated at the 2 hr post-contraction time point relative to REST fibers when normalized to either HSP60 (18.5±7.5 fold) or cardiac -actin (14.7±4.3 fold), although not at 1 or 4 hours. These data indicate that: 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed; 2) HSP60 and cardiac -actin are suitable endogenous normalizing genes in skeletal muscle following contractions; 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 hours after a single bout of fatiguing contractions despite minimal temperature changes and without influence from extracellular sources.