BACKGROUND The sperm chromatin structure assay (SCSA) is a valuable tool for prediction of fertility in vivo, with DNA fragmentation index (DFI) of 30% as a clinically useful cut-off level. Previous studies on fertile men have shown a high level of repeatability, with an intra-individual variability in DFI of ≈ 10%. However, conflicting data on how much the DFI fluctuates within individuals exist. The aim of the present study was to investigate the intra-individual variation of DFI in order to further evaluate the clinical use of SCSA. METHODS Among 2409 consecutive men under infertility investigation, repeated SCSA analyses were performed on 616 samples from men between 18 and 66 years of age. The coefficient of variation (CV) for DFI was calculated. For each patient, we also analyzed whether the DFI value in tests I and II switched the category from <30 to >30%, or vice versa. RESULTS Mean CV for DFI for men with at least two SCSA analyses within a 30-month period was 30.1% (SD 21.5). Compared with the first test, 85% (95% confidence interval: 82-87%) of the men remained on the same side of the cut-off point of 30%. CONCLUSIONS Despite showing a high intra-individual CV for DFI, 85% of the men from infertile couples did not change category between tests, with respect to the cut-off level of 30%. Thus, using the previously established DFI cut-off value of 30%, a single SCSA analysis has a high predictive value for assessing fertility in vivo.