Skeletal muscle is a primary tissue responsible for both the exercise capacity and insulin-induced glucose uptake. Exercise-evoked proteomic alternations in muscle of subjects with prediabetes are of great importance for the elucidation of the connections between insulin resistance and physical exercise. To envision best aviable approach, we compared various ion-chromatogram libraries assembled with the use of off-line high-pH fractionation (HpH), gas-phase fractionation (GPF) and library-less DirectDIATM approach in LC/MS/HRMS DIA proteomic analysis of muscle from normoglycemic (NGT) and prediabetic (IGT) subjects after 3 months of supervised, mixed-mode exercise. Although HpH approach to library generation resulted in best proteome coverage, quantitative and qualitative analysis of HeLa standards, mixed samples and whole study set revealed, that GPF-fractionated hybrid DDA/DIA-acquired libraries yielded best overall balance between the speed of preparation, data collection, data analysis and identification in real-life clinical samples.