Objectives: Many schools internationally are replacing traditional classrooms (TC) with innovative flexible learning spaces (FLS) to improve academic outcomes. Via a stealth approach, there may be additional unintended health benefits if students reduce their total and prolonged sitting. The aim of this study was to compare student sitting patterns between TC and FLS. Design: School-based cross-over trial. Methods: Students at nine secondary schools (n = 191, M age = 13.2 ± 1.0 years) wore activPAL accelerometers in both a traditionally furnished and arranged classroom (TC), and a FLS containing a variety of furniture and layout options, utilizing student-centered pedagogies, for the duration of one double classroom lesson (M = 76 min). The lesson content and teacher were consistent across both conditions. Data were analyzed using multilevel mixed-effects linear regression. Results: In FLS, students spent less class time sitting (mean = 18%; 95% CI: −20.8, −15.0), and accumulated more breaks in sitting (2.1; 95% CI: 0.8, 3.5 per 60 min), more bouts of intermittent (≤9 min) sitting (2.2; 95% CI: 0.8, 3.6 per 60 min), and fewer bouts of prolonged (≤30 min) sitting (−0.2; 95% CI: −0.3, −0.1 per 60 min), than in TC. Students also spent more class time standing (15%; 95% CI: 12.7, 18.0) and stepping (3%; 95% CI: 2.0, 3.1) in FLS than TC. Conclusion: The results suggest that, by stealth, elements of FLS including a variety of furniture and resources, and greater use of student-centered pedagogies, facilitate improvements in adolescents’ sedentary profiles during class time. This may translate into beneficial health impacts over a longer period given the health benefits of reducing total and breaking up prolonged sitting.