Testicular samples obtained for fertility preservation often need to be transported between clinics. This study aimed to mimic this short-term hypothermic storage (4–8°C) and explore the impact of these conditions and the transport medium composition on prepubertal rat testicular tissue samples. Testicular tissue samples obtained from 7 dayspost-partumrats were transferred to six compositionally different basal culture media and a balanced salt solution, which had been kept at 4–8°C prior to transfer. The samples were preserved for either 12 or 24 h in these hypothermic conditions. The potential effects of the short-term storage were evaluated by assessing the morphology, measuring the testosterone levels by radioimmunoassay and analysing 96 genes with TaqMan Low-Density Arrays. Levels of gene expression related to energy, apoptosis, and angiogenesis pathways were altered after hypothermic storage for 12 and especially 24 h. We observed only minor differences in gene expression profiles for germ and testicular somatic cells and no differences in tissue morphology and testosterone production levels. Short-term hypothermic storage of testicular tissue with a maximum duration of 24 h does not affect the overall expression profile of testicular cell-specific genes; however, in a minor way, it affects the expression of specific cellular genes.Lay summaryMale fertility depends on the proper functioning of cells which develop into reproductive cells. Due to an increasing number of childhood cancer survivors suffering from treatment-related fertility problems, as well as recent reports showing a dramatic decrease in sperm counts over the last decades, male fertility preservation has become an important research topic. To date, there is no method to restore fertility for men who are not able to produce sperm. One promising method to preserve the potential fertility of these patients is freezing tissue or cells from the testicles for future fertility treatments. A critical phase in freezing testicular tissue or cells is the time between removing the tissue from the testicles and freezing it. To better understand the impact of this phase on the quality of the testicular tissue, we used the testes of rats as a model for our research. We found that cooling testis tissue has only minor effects on the expression of genes that are important for testis function.