Hair accumulates cortisol over time and concentrations during intervals (IT) are used to assess chronic exposure of animals to situations that increase cortisol secretion. Our objectives were to assess impact of milk yield genotype on hair cortisol when unique unselected (stable milk yield since 1964; n = 12) and contemporary (n = 12) Holsteins that differed in milk yield by more than 4,500 kg milk/305 d were subjected to stressors during IT of the periparturient period and early lactation. Cows were blocked by expected calving date and hair removed from both shoulders and left side 4 wk before expected calving. Shoulder hair was collected at 2 d (IT1), 7 wk (IT2) and 14 wk (IT3) and left side hair at 14 wk (IT4) of lactation. Liver, mammary and tailhead adipose samples were collected at −12 ± 1 DIM (during IT1) and at 4, 14, and 42 ± 1 DIM (during IT2). During IT3, saline or LPS (0.25 μg/kg BW, Escherichia coli 055:B5) was administered twice by iv injections 4 d apart and liver sampled at 0, 4, and 24 h after each injection. Left side samples represented the entire 18 wk study (IT4). Cortisol in washed, ground hair was extracted with methanol and measured by a validated ELISA. Right and left shoulder values were averaged. The IT1 and IT2 samples were assessed for effect of genotype, interval, and interaction by repeated measures using PROC MIXED (SAS) with IT as the repeated effect. The IT3 and IT4 samples were analyzed separately to assess effect of genotype, treatment (saline, LPS) and interaction. Means differed when P < 0.05. There was no interaction in either model. Results indicate hair cortisol accumulation did not differ between genotypes (2.90 ± 0.24 pg/mg) but was increased when cows were in early lactation and subjected to 3 multiple biopsies (IT1 vs. IT2; 2.41 vs. 3.39 ± 0.26 pg/mg; P = 0.002). During IT3 and IT4, there was no effect of genotype and the short duration of the LPS-induced response was insufficient to elicit a change in hair cortisol. Although there is concern that increased milk yield increases stress in cows, it had no effect on hair cortisol concentrations in this study.