The aim or this study was to determine the relationship between non-esterified fatty acids and biomarkers from an automatic milking system (AMS). Fresh dairy cows (n = 102) were selected and milked in Lely Astronaut® A3 milking robots. The rumination time (RT), body weight (BW), milk content and composition parameters, milk fat/protein ratio (F/P), and milk electrical conductivity were registered by the same milking robots. For examining non-esterified fatty acids (NEFAs), blood samples were acquired from cows in the dry period. According to the NEFA concentrations, all cows were divided into two groups: Group I, with <0.300 mEq/L (n = 66), and Group II, with ≥0.300 mEq/L (n = 36). Albumin (ALB), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and cortisol concentrations were also analyzed once a week up to 30 days in milking. The study revealed that the cows in Group I had higher concentrations of ALB, cortisol, and GGT, but the average concentration of AST was lower. In Group 1, the milk F/P was higher, but the milk yield was lower. We hypothesize that biomarkers from AMS could help in the early diagnosis of metabolic diseases after calving or to control negative energy balance before calving.