The aim of the current study was to evaluate the relationship of different parameters from an automatic milking system (AMS) with the pregnancy status of multiparous cows at first service and to assess the accuracy of such a follow-up with regard to blood parameters. Before the insemination of cows, blood samples for measuring biochemical indices were taken from the coccygeal vessels and the concentrations of blood serum albumin (ALB), cortisol, non-esterified fatty acids (NEFA) and the activities of aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were determined. From oestrus day to seven days after oestrus, the following parameters were registered: milk yield (MY), electric milk conductivity, lactate dehydrogenase (LDH) and beta-hydroxybutyric acid (BHB). The pregnancy status was evaluated using ultrasound "Easy scan" 30-35 days after insemination. Cows were grouped by reproductive status: PG- (non-pregnant; n = 48) and PG+ (pregnant; n = 44). The BHB level in PG- cows was 1.2 times higher (p < 0.005). The electrical conductivity of milk was statistically significantly higher in all quarters of PG- cows (1.07 times) than of PG+ cows (p < 0.05). The arithmetic mean of blood GGT was 1.61 times higher in PG- cows and the NEFA value 1.23 times higher (p < 0.05) compared with the PG+ group. The liver function was affected, the average ALB of PG- cows was 1.19 times lower (p < 0.05) and the AST activity was 1.16 times lower (p < 0.05) compared with PG+ cows. The non-pregnant group had a negative energy balance demonstrated by high in-line milk BHB and high blood NEFA concentrations. We found a greater number of cows with cortisol >0.0.75 mg/dL in the non-pregnant group. A higher milk electrical conductivity in the non-pregnant cows pointed towards a greater risk of mastitis while higher GGT activities together with lower albumin concentrations indicated that the cows were more affected by oxidative stress.