Colostrum has a different composition than milk in established lactation. This difference is in part due to the partially open blood-milk barrier, which prevents the interdiffusion of blood and milk components. In the first days of lactation, α-lactalbumin (LALBA), a milk protein, is typically present in blood and several blood-derived proteins are present in milk such as IgG₁ (very high concentration), IgG₂, serum albumin (ALB), and lactate dehydrogenase (LDH). With the exception of IgG₁, which is transferred by active transcellular transport, other proteins are thought to pass paracellularly through the temporarily open barrier. This study aimed to examine the decline patterns of each protein relative to IgG₁, to distinguish between paracellular and transcellular transport through the blood-milk barrier during the first days of lactation. Ten Holstein cows were milked at 4 h after parturition, the next 5 consecutive milkings, and the afternoon milking on d 5, 8, 10, and 14 of lactation for a total of 10 milking time points and blood samples were taken in parallel. Blood and milk samples were analyzed for the concentrations of LDH, ALB, IgG₁, IgG₂ and LALBA. Protein concentration curves were generated from all 10 time points and were evaluated using the tau time constant model to determine the rate of decline of the slope of each protein. When examining blood-derived proteins in milk, the concentration of IgG₁ declined significantly faster than the proteins IgG₂ and LDH. Interestingly, the decline of ALB was not statistically different from IgG₁ nor IgG₂ and LDH. IgG₁ concentration in milk far exceeded levels in plasma, and this protein exhibited a recovery increase in plasma during the experimental period. IgG₂, ALB, and LDH concentrations in milk did not reach plasma levels. Plasma LALBA followed a different pattern, declining significantly slower than all blood-derived proteins in milk. These results indicate that there is active transport of only IgG₁, with a sharp decline at parturition, compared with IgG₂, ALB, LDH, and LALBA which are following the closure of the blood-milk barrier.