In this study, we compared the effects of a 72-h exercise abstinence period and normal exercise on the affect and body dissatisfaction of 58 healthy women (mean age 26.1 years, s = 8.2) who exercised at least four times per week, and explored the relationship between the changes in affect and body dissatisfaction. Participants completed the UWIST-MACL and Body Dissatisfaction Scale to assess affect and body image, and were randomized to an abstinence or control group. Affect and body image for both groups were re-assessed following the 72-h abstinence period. Compared with the control group, the abstinence group had a significant decrease in hedonic tone and energetic arousal, and a significant increase in tense arousal and body dissatisfaction from pre-intervention to post-intervention. There were no significant relationships between the changes in body dissatisfaction and the components of affect. A 72-h exercise abstinence period resulted in increased affect disturbance and body dissatisfaction in regularly active women.