Adolescents spend large amounts of time sitting at school. Little is known about the impact of reducing and breaking-up prolonged sitting during school lessons on adolescents' health. This study aimed to investigate the impact of an intervention to reduce classroom sitting time on adolescents' energy expenditure (EE; kcal/lesson), body mass index (BMI), waist circumference (WC), and musculoskeletal discomfort. A secondary school classroom was equipped with height-adjustable desks, posters promoting the health benefits of and strategies for breaking-up sitting time, and desk stickers reminding students to periodically stand up. Classroom teachers participated in a professional development session. Using a quasi-experimental design, differences between 49 participants who utilised the intervention classroom 2-5 times/week and a comparison group (39 adolescents, matched by year level and subject) who used traditional classrooms, were examined. EE, BMI and WC were objectively measured and musculoskeletal discomfort was self-reported at baseline, 4-weeks, and 17-weeks. Hierarchical linear and multilevel logistic regression-mixed models were used to examine intervention effects, adjusting for baseline values, sex and age. EE was significantly higher at 4-weeks and 17-weeks (29.4 and 37.7 kcal/lesson, respectively), BMI was higher at 4-weeks (0.34 kg/m2), and WC was lower at 4-weeks and 17-weeks (-3.53 and -2.64 cm, respectively) in the intervention compared to the comparison group. No intervention effect was found for musculoskeletal discomfort. Findings provide preliminary indications that these strategies may benefit health among adolescents in the short term. However, extended longer-duration trials are needed to determine longer-term health effects.