Background As a potential indicator of strain, fatigue is an important outcome in occupational health research. The current study examined the influence of positive (eustress) and negative (distress) work experiences on changes in fatigue from morning to at‐work. It was expected that within‐person changes in fatigue from waking to working would be moderated by employees' experiences of stress, pain, happiness, and meaningfulness at work. Methods Data on 1,195 full‐time working adults were collected through the Bureau of Labor Statistics' 2010 American Time Use Study (ATUS) using a day reconstruction method to assess fatigue at two time points (morning and during work) and employees' eustress and distress experiences during work. Results Multilevel modeling showed that the indicators of distress, stress and pain, predicted higher morning fatigue and stronger increases in fatigue during the workday. The indicators of eustress, happiness and meaningfulness, predicted lower fatigue at both time points but not temporal changes. Conclusions These results contribute to understanding changes in employees' fatigue and suggest that the differential effects of distress and eustress experiences at work may be important to consider in fatigue management interventions.