When evaluating trace DNA recovered from evidential items in forensic casework, it is crucial to consider how the DNA got there, and such evaluative interpretations should ideally be informed by published experimental data. A key activity-level question is whether the DNA obtained comes from the regular user, the last user (ostensibly the user at the time of the crime) or from indirect transfer events. The aim of this experiment was to provide data to contribute to answering this question, particularly when considering opportunistic crimes, in which an offender might grab the nearest item at hand required for their purpose, e.g. a weapon or tool, and therefore only handle it very briefly. Volunteers ('regular users') used knives in a prescribed manner to simulate regular use (one user per knife); DNA recovery by mini-tapes from these knives gave ˜1-10 ng DNA, with <16% non-donor DNA from indirect transfer events. Different volunteers ('second users') then stabbed replicate sets of regularly-used knives into a foam block for either 2, 30 or 60 s (on different occasions), with each timeframe in triplicate, and DNA was recovered from the knife handles using mini-tapes. For knives regularly-used by three of the four volunteers, the ratios of regular user to second user DNA were approximately 4:1, 2:1 and 1:1 for durations of use by the second user of 2, 30 and 60 s, respectively. Analysis of the respective quantities of DNA showed that this trend resulted from a decrease in regular user DNA via transfer to the second user's hands, rather than an increase in DNA deposition from the second user. However, for knives regularly-used by the fourth volunteer, DNA from the regular user remained at significantly higher quantities than DNA from the second user and unknown sources, irrespective of duration of use by the second user. Furthermore, one volunteer deposited a similar amount of DNA through regular use as the amount of indirectly-transferred unknown DNA deposited by another volunteer's hands. These observations indicate that caution should be taken when relying solely on absolute quantities of DNA to inform evaluative interpretations, and other parameters, such as profile quality and relative contributions to mixed profiles, should also be taken into account. To better assist activity level assessments, more extensive studies of this manner should be conducted to obtain probability distributions of different types of profiles resulting from this kind of activity.