Introduction: Immune therapy plays a larger role in cancer treatment these days, but in order to find new therapies and improve already existing ones, more knowledge about the immune system is needed. The peripheral blood contains many different cell types, some extensively studied, some less well-known. By using multicolor flow cytometry, the immune status of an individual can be displayed in relatively short time. Aim: The aim of this study was to develop a multicolor flow cytometry method in order to examine the distribution of 31 cell types, with a focus on immune regulatory cells, in peripheral blood in healthy donors, as well as examine the impact of cryopreservation on the different subsets. Methods: After isolating the mononuclear cells from peripheral blood using Ficoll separation, each sample (n = 19) were analyzed with three flow cytometry panels. The remaining cells were cryopreserved in -190°C and later thawed and analyzed the same way as the fresh samples were. Results: The cell distribution in fresh samples were mostly consistent with other studies. While the percentage of many cell types remained unchanged after thawing, the total percentage of T helper cells and some subsets were decreased in frozen samples, leading to a decrease in total T cells. Furthermore, the percentage of total monocytes were increased and the distribution of monocyte subsets were altered in frozen samples, among others. Conclusion: This study confirms the results of other studies of the human immune system and provides valuable knowledge about the impact of cryopreservation.