Our objective was to evaluate the effect serial corpus luteum (CL) biopsy collection on circulating progesterone concentrations and corpus luteum size, blood flow, and gene expression in pregnant cows. Pregnant primiparous cows on d 27 of gestation with a single noncavitary CL were assigned randomly to a biopsy (BIO, n = 8) or sham biopsy (SB, n = 8) treatment. Biopsies were collected with an automatic biopsy needle guided through transvaginal ultrasonography (US). A CL biopsy was collected every 48 h (5 total) from d 27 to 35 in BIO cows, whereas the sham biopsy procedure was performed in SB cows. Blood samples were collected at the time of biopsy collection and 10 d after the last biopsy to measure progesterone (P4) concentration. Immediately after CL biopsy or sham biopsy and 45 d after AI, B mode and Power Doppler transrectal US were performed to evaluate luteal size, blood flow, and pregnancy viability. The mRNA abundance of genes associated with steroidogenesis (3βHSD and STAR) inflammation (TNFa), and apoptosis (TNFa, BCL2, and BAX) was determined by RT-qPCR. Genes of interest were normalized against 3 stable reference genes (GAPDH, H3F3B, and RPS9). Continuous variables were analyzed by ANOVA with repeated measures using the Mixed procedure of SAS. Embryo loss ocurred in 3 BIO (38%) and one SB cow (13%) between d 27 and 45. There was no effect (P = 0.84) of treatment (TRT), time (TIM; P = 0.83) or TRTxTIM (P = 0.15) on P4 concentration (ng/mL) from d 27 to 45 (BIO = 1.70 ± 0.08; SB = 1.72 ± 0.08). Total CL area tended (P = 0.05) to be greater for cows in SB (586.2 ± 16.4 mm²) than BIO (530.6 ± 11.7 mm²) but was not affected by TIM (P = 0.87) or TRT×TIM (P = 0.46). Area of CL with blood flow (BIO = 44.2 ± 2.5%; SB = 38.9 ± 2.5%) was not affected by TRT (P = 0.66), TIM (P = 0.14), or TRT×TIM (P = 0.3). There was no effect (P > 0.10) of TRT, TIM, or TR×TIM on mRNA relative abundance for any of the genes evaluated. We conclude that collecting multiple CL biopsies (up to 5) every 48 h from d 27 to 35 of pregnancy did not cause mayor alterations to CL function as determined by progesterone secretion and CL size, blood flow, and gene expression. Supported by USDA Hatch Multistate project NYC 127813.