No previous study has used proteomics to investigate the effects of exercise training on human skeletal muscle. Five recreationally active men completed a 6-wk training programme involving three sessions per week, utilising six 1-min bouts at maximum oxygen uptake (V O(2)max) interspersed with 4 min at 50% V O(2)max. Vastus lateralis was biopsied at standardised times before and after the training intervention. Protein expression profiling was performed using differential analysis of 2-DE gels; complemented with quantitative analysis (iTRAQ) of tryptic peptides from 1-DE gel lane-segments using LC-MALDI MS/MS. Interval training increased average V O(2)max (7%; p<0.001) and was associated with greater expression of mitochondrial components, including succinate dehydrogenase, trifunctional protein-alpha and ATP synthase alpha- and beta-chains. 2-DE resolved 256 spots, and paired t-tests identified 20 significant differences in expression (false discovery rate <10%). Each differentially expressed gene product was present as multiple isoelectric species. Therefore, the differences in spot expression represent changes in post-transcriptional or post-translational processing. In particular, modulation of muscle creatine kinase and troponin T were prominent. Pro-Q Diamond staining revealed these changes in expression were associated with phosphorylated protein species, which provides novel information regarding muscle adaptation to interval training.