Abstract The objective was to determine associations between behavior during head-lock restraint and reproductive performance in dairy heifers. Behavior of 817 Holstein heifers from four farms was evaluated at feeding (Days 0 and 7) while restrained in a self-locking stanchion. All heifers were assigned a body condition score (BCS; 1, emaciated to 5, obese) on Day 0. Heifers were timed-inseminated on a maximum of three occasions to determine impact of behavior for first service pregnancy per AI (FS-P/AI) and cumulative P/AI (C-P/AI). Ovulation was synchronized with an Ovsynch synchronization protocol for first service and thereafter either Ovsynch and/or prostaglandin F 2α -based protocols. More heifers displayed calm escape behavior (P < 0.05) compared with mild or aggressive escape behaviors (45.2, 28.2 and 26.6%, respectively). Adjusting for BCS (P < 0.05), FS-P/AI was greater (P < 0.05) for calm heifers compared with aggressive escape behavior, 58.0% (214/369) vs 48.2% (105/218), with FS-P/AI of heifers with mild aggressive behavior [53.5% (123/230)] intermediate and did not differ from other means. Adjusting for BCS (P < 0.0001), C-P/AI was greater (P < 0.0001) for heifers with calm compared with mild or aggressive escape behaviors [84.8% (313/369), 71.3% (164/230) and 64.7% (141/218), respectively]. Serum cortisol concentrations were not different among behavior categories, but serum substance P concentrations were greater (P < 0.05) in aggressive heifers compared with mild or calm heifers, 97.1 ± 4.9, 58.4 ± 2.9 and 52.3 ± 2.6 ng/mL, respectively. In conclusion, Holstein heifers with aggressive escape behavior during head-lock restraint had significantly reduced reproductive performance. Highlights • During headlock restraint, more heifers showed calm escape behavior. • Mean serum cortisol concentrations did not differ among heifers with aggressive, mild and calm escape behavior. • Mean serum substance P concentrations was greater in heifers that expressed aggressive compared with mild and calm escape behavior. • First service pregnancy/AI was greater in heifers that expressed calm compared with aggressive escape behavior. • Cumulative pregnancy/AI was greater for heifers that expressed calm compared with mild and aggressive escape behavior. [ABSTRACT FROM AUTHOR]