Background: This study analyzed the effect of walking breaks or low-volume high-intensity interval exercise (LV-HIIE) on markers of metabolic syndrome relative to a day of prolonged sitting. Methods: Twenty-five adults with excess body fat participated in this crossover trial: (1) 10-hour sitting day (SIT), (2) LV-HIIE followed by a sitting day (EX+SIT), and (3) sitting day with 5-minute walking breaks for every 20 minutes (SIT+WB). Glucose and blood pressure (BP) were measured before and 1 hour after 4 meals and 2 hours after lunch. Triglycerides were measured at baseline, 2, and 3.5 hours after lunch. Generalized mixed models were used to identify differences in the area under the curve (AUC) of BP and incremental AUC (iAUC) of glucose and triglycerides among the sessions. Results: iAUC-glucose was lower in SIT+WB than SIT (β = −35.3 mg/dL·10 h; 95% confidence interval, −52.5 to −8.2). AUC-diastolic BP was lower in SIT+WB than SIT (β = −14.1 mm Hg·10 h; 95% confidence interval, −26.5 to −1.6) and EX+SIT (β = −14.5 mm Hg·10 h; 95% confidence interval, −26.9 to −2.1). There were no differences in triglycerides and systolic BP levels among the sessions. Conclusion: Adults with excess body fat present lower glucose and diastolic BP during a day with breaks in sitting time compared with a prolonged sitting day with or without an LV-HIIE session.