Objectives Successful implementation of asymptomatic testing programmes using lateral flow tests (LFTs) depends on several factors, including feasibility, acceptability and how people act on test results. We aimed to examine experiences of university students and staff of regular asymptomatic self-testing using LFTs, and their subsequent behaviours. Design and setting A qualitative study using semistructured remote interviews and qualitative survey responses, which were analysed thematically. Participants People who were participating in weekly testing feasibility study, between October 2020 and January 2021, at the University of Oxford. Results We interviewed 18 and surveyed 214 participants. Participants were motivated to regularly self-test as they wanted to know whether or not they were infected with SARS-CoV-2. Most reported that a negative test result did not change their behaviour, but it did provide them with reassurance to engage with permitted activities. In contrast, some participants reported making decisions about visiting other people because they felt reassured by a negative test result. Participants valued the training but some still doubted their ability to carry out the test. Participants were concerned about safety of attending test sites with lots of people and reported home testing was most convenient. Conclusions Clear messages highlighting the benefits of regular testing for family, friends and society in identifying asymptomatic cases are needed. This should be coupled with transparent communication about the accuracy of LFTs and how to act on either a positive or negative result. Concerns about safety, convenience of testing and ability to do tests need to be addressed to ensure successful scaling up of asymptomatic testing.