Several studies have examined the rate of backfat mobilization in periparturient dairy cattle, but the rate of intramuscular fat (IMF) mobilization relative to backfat mobilization and muscle tissue mobilization does not appear to have been studied. Our objective was therefore to characterize the changes in IMF relative to changes in muscle thickness and backfat thickness (BFT) in the physiologic response to negative energy balance in periparturient dairy cattle. One hundred and six periparturient Holstein-Friesian cattle (34 primiparous, 72 multiparous) at -3 and +28 days relative to parturition were examined. A 5 MHz linear ultrasound probe was used to measure BFT and the maximum thickness and IMF percentage (determined using a thresholding-segmentation method) of the longissimus dorsi muscle in the thoracic region (LD-thoracic). Spearman’s correlation coefficient (rs) and mixed models analysis were used for statistical analysis and P < 0.05 was declared significant. The mean decrease in BFT over the 31 day period was similar for primiparous (-46%) and multiparous cows (-45%). The mean decrease in IMF of the LD-thoracic muscle was similar for primiparous (-22%) and multiparous cows (-30%). Likewise, the mean decrease in LD-thoracic muscle thickness was similar for primiparous (18%) and multiparous cows (21%). The decrease in IMF was not associated with BFT decrease (rs=0.03), but was associated with the decrease in LD-thoracic muscle thickness (rs=0.59, P=0.0011). We conclude that ultrasonographic measurement of the LD-thoracic muscle thickness and IMF complements ultrasonographic measurement of BFT when quantifying negative energy balance in periparturient dairy cattle.