Research has shown that milking frequency influences milk production in dairy cows. In automatic milking system (AMS) farms, not only milking frequency per cow varies, but also their milking interval. The objective of this study was to evaluate the association between milking interval and cow milk production, milk fat percent, and milk protein percent in AMS farms. We used one year of visit-level data from the AMS software (summer 2017 to summer 2018; 4,978,495 visits), from 36 free-flow AMS (Lely Astronaut, Lely, the Netherlands) farms in Minnesota and Wisconsin. The MIXED procedure of SAS 9.4 (SAS Institute, Inc., Cary, NC) was used to analyze the data. Milk production/visit, milk fat, or milk protein were the outcome variables in each of the 3 separate models. The main predictor of interest was milking interval. Days in milk (DIM), concentrate intake/visit, milking speed, and milking time were added to the model as covariates. Cow within farm was used as random effect. Milk production/visit had a median value of 12.7 (±4.51) kg. The average for milk fat was 3.78 (±0.76) %, and for milk protein 3.08 (±0.25) %. Milking interval had a median of 472.8 (±180.2) min. The median DIM was 146.0 (±106.1) d. Concentrate intake had a median of 1.69 (±0.58) kg/visit. Median for milking speed was 2.97 (±1.13) kg/min, and for milking time it was 4.32 (2.08) min. For each hour increase in milking interval, milk production increased by 0.19 kg/visit (P < 0.0001), fat increased by 0.02 percentage units (P < 0.0001), and protein increased by 0.03 percentage units (P < 0.0001). Milking interval appears to influence milk production/visit and milk components. However, further research investigating this association is warranted.