Aims/hypothesis Polycystic ovary syndrome (PCOS) is an insulin resistant (IR) state. Increased skeletal muscle lipid content and impaired mitochondrial biogenesis have beenimplicated in the pathogenesis of IR. We investigated whether differences in these variables explain the IR of women affected by PCOS and whether improvements in IR with exercise are reflected by changes in these variables. Methods Sixteen PCOS and 13 non-PCOS overweight women were assessed, and eight PCOS and seven non- PCOS women were reassessed after 12 weeks of moderate and vigorous exercise training. Outcomes included insulin sensitivity (glucose infusion rate [GIR]), skeletal muscle gene expression and protein abundance, enzyme activity of selected mitochondrial components, and computed tomography (CT) attenuation-estimated muscle lipid. Results GIR was lower in women with PCOS versus those without (p00.01) and increased with exercise in both groups. Baseline CT muscle attenuation suggested a trend to less muscle lipid in PCOS, which increased with exercise training, with a difference in the change in muscle lipid (p00.01, age-corrected), compared with non-PCOS women. GIR correlated with PGC1A gene expression across the whole group; skeletal muscle expression of mitochondrial biogenesis markers was not different between groups at baseline, or after training. Neither lipid changes nor mitochondrial changes correlated with changes in GIR. Conclusions/interpretation Differences in IR in women with and without PCOS were not explained by differences in skeletal muscle lipid ormitochondrial parameters. Improvements in IR with exercise were dissociated from mitochondrial parameters. CT muscle attenuation suggested a differential capacity f PCOS muscle to store lipid compared with non-PCOS.