Microsatellites are highly unstable regions in the human genome. If mutations occur in the germline they might be transmitted to the offspring and therefore bear a high risk of influenc-ing future generations. In order to find out about the molecular mechanisms of such muta-tions, we studied the mutation rates of 3 microsatellites in sperm cells of both younger and older men. According to the paternal age effect, microsatellite mutation rates should increase with age. We are the first group to use Duplex Sequencing for the analysis of microsatellites in the male germline. This ultra-sensitive method has a 10,000-fold higher accuracy than other next-generation sequencing techniques which are commonly used to measure microsatellite muta-tion rates. We optimized a library preparation protocol which should able to generate robust and consistent data. Our results support several previously reported findings of microsatellite mutation features: (1) Mutation rates become higher with increasing length of a microsatellite. (2) Mutations pri-marily affect one repeat unit. (3) Microsatellites which are relatively long tend to decrease in length. (4) Imperfect microsatellites have lower mutation rates than perfect microsatellites. Besides, our data suggest that uninterrupted stretches of imperfect microsatellites behave similar to perfect microsatellites. (5) Our results also indicate that mutation rates were higher in sperm cells of older men than in sperm cells of younger men. However, our data did not allow to make more precise conclusions regarding the question of a possible paternal age effect. In conclusion, we developed a protocol for Duplex Sequencing which is appropriate to meas-ure mutation rates of microsatellites in human sperm cells.