Regular bouts of physical activity may cause changes in gene expression that accumulate over time and ultimately affect phenotypes, such as body weight, blood lipid profile and tumour development. Furthermore, acute activity may affect gene expression and phenotypes differently depending on whether the individual is regularly inactive or active. One-month-old male Sprague-Dawley rats (n = 72) were equally divided into SED (standard laboratory cage, n = 24), PA (large activity box, n = 24) and EX groups (exercise wheel inside standard cage, n = 24). At 3 months of age, half the animals from each group were killed at rest and the other half following 30 min of physical activity. The RNA was extracted from cardiac tissue, and microarray analysis was performed on 27,000 genes. Select gene results were validated using quantitative PCR. No gene expression differences occurred when comparing all 3-month-old groups at rest. A relatively small percentage of genes (1.9%) were differentially expressed (P < 0.05) following acute swimming activity in all groups, but only 37 unique and identifiable genes reached or exceeded twofold differences in expression. The genes Atf3, Fos, Apold1 and Pxdn were expressed differently among SED, PA and EX following acute activity, with a clear separation of the magnitude in gene expression with SED > PA > EX. Differences in gene expression levels in young physically inactive and active animals following acute activity have different regulatory roles in gene networks that affect health-related phenotypes.