The objective was to assess the effect of pre-milking waiting time in the holding area on the subsequent lying behavior and number of steps of dairy cows. Holstein cows (parity 1, n = 17; parity ≥ 2, n = 111) from a dairy in northern Colorado were affixed with a pedometer (IceQube, IceRobotics, Edinburgh, UK) in one rear leg providing number of steps (STP), lying time (LY; min), and standing time (STD; min). Cows were milked 3× in a 60-stalls rotary milking parlor (DeLaval). Time of entrance to the milking stall for each individual cow was automatically recorded from January to June 2018 (average DIM at enrollment = 15.3 ± 7.1). Starting time for each milking pen was also available. Individual waiting time was calculated for each milking as the difference between the pen starting time and the time of cow entrance to the milking stall. Waiting times were categorized (WaitCat) in low (≤30 min) and high (>30 min). STP, STD, and LY were added until the beginning of the subsequent milking and standardized in minutes/hour. General linear models were created to test the associations between waiting time categories and activity parameters. Parity (1; ≥ 2), shift (morning, afternoon, night), and their interactions with WaitCat were also included. Table 1 presents activity values by waiting category, as well as P-values for variables included in the models. Our preliminary analyses determined a tendency toward significance for the effect of WaitCat and parity on STP. A significant interaction between shift and Waitcat was observed for all the outcomes, suggesting that daily activities may modify the effect of waiting time on the behavior of dairy cows. Further analyses considering other potential confounding factors are required to clarify the significance of pre-milking waiting time on cow behavior. Table 1 (Abstr. 52). Summary of activity parameters (mean ± SE) by pre-milking waiting category

	Steps (no./h)	Standing time (min/h)	Lying time (min/h)
WaitCat (min)
≤30	107.5 ± 4.24	34.8 ± 0.54	25.2 ± 0.54
>30	112.0 ± 4.25	34.7 ± 0.54	25.3 ± 0.54
P-value
Waitcat	0.10	0.70	0.70
Parity	0.002	0.80	0.80
Waitcat × Parity	0.01	0.60	0.60
Shift	<0.001	<0.001	<0.001
Shift × Waitcat	0.60	0.03	0.03