In dairy cattle, mammary biopsies are commonly used to study mammary development and function. The objective of this study was to investigate the behavioral changes following mammary biopsy. Pregnant, nonlactating Holstein dairy cows (n = 9/treatment; 20 d before expected calving date) were exposed to either: 1) a biopsy procedure, in which mammary tissue samples (60 × 4 mm in diameter) were obtained using a biopsy tool from the rear left quarter, following administration of a sedative (xylazine, 20 µg/kg of BW) and local anesthesia (3 mL lidocaine), or 2) a sham procedure, in which cows were removed from the pen and restrained for a similar duration of time as for the biopsy procedure. Cows were fitted with accelerometers to record daily standing time, standing bout frequency, and lying side. Dry matter intake (DMI) was recorded daily using the Calan gate system. Daily activity data and DMI were recorded for 1 wk following the biopsy or sham procedure, and analyzed in a general linear mixed model with day as a repeated measure. The biopsy procedure had no effect on DMI (11.2 kg/d; SE = 0.67; P = 0.88) or total daily standing time (10.0 h/d; SE = 0.44, P = 0.44). However, relative to control cows, biopsied cows had more frequent standing bouts (11.8 vs. 9.9 bouts/d; SE = 0.59; P = 0.04) of shorter duration (52.2 vs. 64.8 min/bout; SE = 4.4; P = 0.05) with no interaction of treatment and day (P > 0.33). On d 1 following the procedure, biopsied cows also showed greater laterality in their lying behavior (P = 0.021), having longer lying bouts on their right side than left (97.8 vs. 70.0 min/bout; SE = 8.6; P = 0.015) whereas control cows showed no lying side preference (P = 0.56). These results suggest that mammary biopsy does not affect overall activity and feeding behavior, but has subtle effects on activity, which may be indicative of increased restlessness or short-term discomfort in the biopsied quarter. These effects, however, dissipate rapidly following biopsy.