Abstract Objectives Many workers had to switch their standard work from the office to home-based telework during the COVID-19 pandemic. Previous studies reported that telework was associated with increased sickness presenteeism. Several studies have examined the association between lifestyle behaviors and presenteeism among workers. However, there are few studies investigating the relationship among teleworkers. Thus, this study examined the association between daily activities and presenteeism in a longitudinal cohort of teleworkers in Japan. Methods A prospective cohort study with a two-year observation was conducted. The participants were 831 workers belonged to five companies in Japan. For both surveys, self-administered questionnaire surveys on daily activities and presenteeism were conducted. Presenteeism was assessed using the short-form Japanese version of the World Health Organization Health and Work Performance Questionnaire. The data were analyzed using a generalized estimating equation approach, controlling for potential confounders such as age, gender, working hours, position, and health status. Results 742 participants who teleworked in both surveys with no missing answers were analyzed. Among male workers (n = 427), chatting (β = 5.30, 95% CI: 1.52-9.07) and setting daily goals (β = 4.16, 95% CI: 1.52-9.07) were associated with decreased presenteeism. Among female workers, exercise (β = 4.33, 95% CI: 0.95-7.71) was associated with decreased presenteeism. Conclusions Although gender differences were observed, there is preliminary evidence from longitudinal studies that frequent activities such as chatting, setting daily goals, and exercising may protect against presenteeism among teleworkers. Our findings could be used to educate daily telework activities to prevent sickness presenteeism. Key messages • Daily activities during telework may worsen or improve presenteeism. • Social isolation may be an important indicator of presenteeism during telework.