Low-cost workplace interventions are required to reduce prolonged sitting in office workers as this may improve employees’ health and well-being. This study aimed to assess the acceptability and feasibility of an e-health intervention to reduce prolonged sitting among sedentary UK-based office workers. Secondary aims were to describe preliminary changes in employee health, mood and work productivity after using an e-health intervention. Healthy, university office workers (n = 14) completed this study. An 8 week randomised crossover design was used, consisting of two trials: Intervention (computer-based prompts) and Control. Eligibility and retention rates were recorded to assess the feasibility of the trial and interviews were conducted following the intervention to explore its acceptability. Sitting, standing and stepping were objectively assessed prior to and during week 8 of each trial. Before and after each trial, measurements of vascular function, cerebrovascular function, mood and work productivity were obtained. This study had eligibility and retention rates of 54.5% and 77.8%, respectively. Participants expressed a lack of autonomy and disruption to their workflow when using the e-health intervention, raising concerns over its acceptability and long-term implementation. Preliminary data indicate that the intervention may improve the patterning of activity accrued during work hours, with increases in the number of standing and stepping bouts completed, in addition to improving vascular function. This e-health intervention is feasible to deliver in a cohort of university office workers. However, adaptations to its implementation, such as personalised settings, are needed to increase acceptability before larger trials can be conducted.