Background: Flexible work schedules and on-call work are becoming more and more common in working life. However, little is known about the effects of on-call work on health. Methods: Using a daily survey method, 31 employees from an Information Technology Service Organisation filled out a questionnaire four times a week while they were on call and another four times a week while they were not on call. An evaluation of cortisol levels was included. Multilevel analyses were used to evaluate the data. Results: Results showed increases in irritation and negative mood and decreases in social activities, household activities, and low-effort activities. No effects were found concerning the secretion of cortisol. There were no significant differences between those employees who were actually called in to work during the on-call period and those who were not. Conclusions: Flexible work schedules like on-call work have effects on well-being. The mere possibility of being disturbed by calls shows negative consequences, regardless of whether the employees are actually called in or not.