In laboratory mice sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA versus the commonly applied terminal cauda epididymis dissection. The collected sperm samples were analyzed using computer assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymis dissection. Based on computer assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymis dissection. In addition, we found significantly more morphological deformities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilizations, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits.