In forensic investigations, the collection of biological material under fingernails may provide important evidence in cases of physical or sexual assault. Among these, scenarios suggesting alternative activities for the presence/absence of the DNA rather than questioning the donor of the trace are particularly challenging. To provide data supporting the interpretation of these cases, we investigated the transfer, persistence, and presence of background male DNA under female fingernails in controlled experiments of simulated scratching. Unlike previous studies, subungual samples were collected over short and long periods, up to 24 h after the scratching without preliminary cleaning of the nails. Y-STRs data showed that the DNA of the male individual scratched by a woman was detected in fingernail samples collected immediately and up to 6 h post-scratching. A notable decrease in male DNA quantity was observed after the first 3 h of scratching. Interestingly, the same foreign Y-STR profiles, different from the participating individuals, were observed between 6 and 24 h post-simulation. Overall, our data confirm that the detection of the offender's DNA from subungual samples is very likely immediately after the assault; yet, persistent background or newly transferred DNA may challenge the interpretation of traces collected after 6 h. Finally, one scenario was discussed to illustrate the value of these data for evaluating fingernail evidence when considering activity-level propositions.