Proteomic analyses are powerful methods and have the potential to yield vast amounts of data. The available proteomic methods have been hampered by large methodological errors in quantification, due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and the purpose of this investigation was 1) to develop a sample preparation protocol for human skeletal muscle for 2-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one such system, the Ettan™ DIGE. To test repeatability, nine aliquots from the same homogenate were labeled with 3 different CyDye™ dyes (Cy2, Cy3, Cy5). Samples were run on 18 x 24 cm gels, scanned with a Typhoon™ 9410 laser scanner and analyzed in the DeCyder™ software. When selecting spots only appearing in triplicates (N = 1314) the mean error was 1.7 % (SD: 10.5%; 95% CI: 1.1-2.4%). By setting the significance level to 99%, no false positive changes in protein volume ratios were detected. The protocol presented here used only 0.5 mg tissue and allowed the separation of > 2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of < 20% can be detected. The method is especially useful when comparing muscle proteins between different conditions, for example: healthy and diseased tissue, before and after treatment or different exercise protocols