Determination of serum prolactin concentration is used to diagnose hyperprolactinemia; however, it should be kept in mind that prolactin levels are affected by several preanalytical factors, such as circadian rhythm (prolactin levels are highest during the night and immediately after waking up), stress, breastfeeding, physical activity and drugs (antipsychotics, estrogens, tricyclic antidepressants). According to the most of studies, physical activity instantly increases the concentration of prolactin. This study aimed to examine whether there was a statistically significant difference in the prolactin concentration immediately after physical activity in comparison with the prolactin concentrations after 20 or 30 minutes of rest. The aim was also to determine the significance of the difference in repeated measurements of prolactin concentration after 20 minutes of rest compared to prolactin concentration after 30 minutes of rest. 21 adult respondents voluntarily participated in the study. Participants were asked to walk for 15-30 minutes or come by bicycle before arriving for a blood sampling. Upon arrival, the blood was drawn for three times: the first time was immediately upon arrival, the second time was after 20 minutes of rest, and the third time was after 30 minutes of rest. The sandwich electrochemiluminescence method (ECLIA) was used for determination of the serum prolactin concentrations. Statistically significant differences in the concentrations of prolactin were determined with the Wilcoxon signed - rank test. The concentration of prolactin immediately after physical activity was found to be higher than the concentrations of prolactin after 20 and 30 minutes of rest. Further results showed that resting for 20 minutes contributed to the stabilization of prolactin concentration, which was statistically significantly different from the prolactin concentration after 30 minutes of resting. However, as medians of prolactin concentrations were almost equal and were not clinically significantly different, we could conclude that the two resting protocols caused an identical preanalytical effect and can be applied equally. Considering the obtained results, which showed elevated prolactin concentrations at the first measurement point (immediately upon arrival to the laboratory) compared with the post-resting concentrations, we conclude that the whole resting procedure before blood sampling for determination of prolactin concentration is justified and that physical activity causes falsely elevated results.