Digital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten month period of exercise training. The study cohort comprised 7 thoroughbred racehorses from a single training yard. Skeletal muscle biopsies were collected at rest from the gluteus medius at two time points: T1 (unconditioned), (9 +/- 0.5 months old) and T2 (conditioned) (20 +/- 0.7 months old). The most highly abundant genes in the muscle transcriptome were those involved in muscle contraction, aerobic respiration and mitochondrial function. A previously unreported over-representation of genes relating to RNA processing, the stress response and proteolysis was observed. Following training 92 tags were differentially expressed of which 74 were annotated. Sixteen genes showed increased expression, including the mitochondrial genes, ACADVL, MRPS21 and SLC25A29. Among the 58 genes with deceased expression MSTN, a negative regulator of muscle growth had the greatest decrease. Functional analysis of all expressed genes using FatiScan revealed an asymmetric distribution of 482 Gene Ontology groups and 18 KEGG pathways. Functional groups with highly significantly (P < 0.0001) increased expression included mitochondrion, oxidative phosphorylation and fatty acid metabolism while functional groups with decreased expression were mainly associated with structural genes and included the sarcoplasm, laminin complex and cytoskeleton. Examination of muscle expression changes in 7 thoroughbred horses following 10 months of exercise training using digital gene expression with NlaIII.