This study aimed to identify the emotional/affective sources of discrepancies between physical activity behavior and a widely used self-perception measure of physical activity motivation. Overweight women (body mass index [BMI] ≥ 25 kg/m2, 18-64 years of age; N=37) were recruited from Arizona State University community through flyers and online newsletters. Participants wore a SenseWear accelerometer for 6 nights and 7 days and followed their normal patterns of daily living. Participants then completed a single lab visit and verbally responded to questions from the Behavorial Regulation Exercise Questionnaire (BREQ-2) while being video and audio recorded. Captured emotional responses were evaluated with facial recognition software (Noldus FaceReader). Discrepancies between BREQ-2 responses and physical activity behavior were associated with happiness and sadness emotional responses extracted from the facial recognition software using regression-based analyses. Results indicated an association between monitored physical activities and captured emotional response - specifically sadness - and that as intensity in physical activity increases, motivation increases. Associations between happiness/sadness and physical activity were not observed for all intensities of physical activity. A marginally significant association was observed for amotivation and sedentary, light-intensity physical activity, and moderate-vigorous physical activity in the sample. This study demonstrates a proof-of-concept for the integration of an empirical evaluation of happiness and sadness emotional states into the relationship between physical activity motivation and behavior.