The aim of the study was to observe the effect of longer fasting of dairy cows during lactation on the insulin and thyroxine levels in blood serum. The test was performed at the Research Institute of Animal Production, Nitra. The blood samples for determination of the hormone levels were collected in the morning from the vena jugularis of four fasted 4-5-year-old dairy cows of the Black Pied breed during lactation with an average daily performance of 16.91. The blood was collected on the first, fifth, sixth, seventh, eight and ninth day (fasting) and then on the fifteenth and twenty first day of the test (feeding). The insulin level in the blood serum of fasted dairy cows was more or less balanced till the seventh day of fasting, and ranged from 4.33 to 4.91-mu-U.ml-1. On the last two days of fasting, the insulin level increased up to 7.11-mu-U.ml-1 on the 9th day of fasting. The insulin level was significantly higher during feeding than during starvation. The thyroxine level was statistically significantly lower in fasted dairy cows. Increasing insulin level at the last two collections of the fasting period was probably closely connected with the shifting of the endocrine balance from dominant position of insulin to the dominance of the growth hormone and glucocorticoids. A comparatively high effect on the increase of the insulin level was exerted during that time also by the increasing amounts of non-esterified fatty acids. Starvation significantly reduced the levels of the studied hormones in dairy cows during lactation.