18Swim-up is a sperm purification method that is being used daily in andrology labs around the 19 world as a simple step for in vitro sperm selection. This method accumulates the most motile 20 sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction but 21 the underlying reasons are not fully understood. In this article, we compare metabolic rate, 22 motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The 23 metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates 24 simultaneously and thereby delivers the metabolic rates in real time. Our study confirms the 25 upper fraction of bull sperm has improved motility compared to the cells in the lower fraction 26 and shows higher metabolic rates. This pattern was consistent across media of two different 27 levels of viscosity. Sperm with longer flagella are selected in the upper fraction. We conclude 28 that the motility-based separation of the swim-up technique is based on metabolic differences. 29Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm 30 quality, which is likely particularly useful in cases of asthenozoospermia and teratospermia. 31Furthermore, metabolic measurements of sperm cells can reveal differences in metabolic 32 pathways in different environments. 33 2 1 Keywords 2 swim-up, bovine sperm, metabolism, sperm selection, sperm quality 3