Background: A large majority of primary school pupils fail to achieve 30-minutes in-school moderate-to-vigorous physical activity (MVPA). The aim of this study was to investigate MVPA accumulation and subject frequency during academic lesson segments and the broader segmented school day. Methods: 122 children (42.6% boys; 9.9±0.3yrs) from six primary schools in North East England, wore uniaxial accelerometers for eight consecutive days. Subject frequency was assessed by teacher diaries. Multilevel models (children nested within schools) examined significant predictors of MVPA across each school-day segment (lesson one, break, lesson two, lunch, lesson three). Results: Pupils averaged 18.33±8.34 minutes of in-school MVPA and 90.2% failed to achieve the in-school 30-minute MVPA threshold. Across all school-day segments, MVPA accumulation was typically influenced at the individual level. Lesson one and two - dominated by Math and English - were less active than lesson three. Break and lunch were the most active segments. Conclusion: This study breaks new ground, revealing MVPA accumulation and subject frequency varies greatly during different academic lessons. Morning lessons were dominated by the inactive delivery of Math and English, whereas afternoon lessons involved a greater array of subject delivery that resulted in marginally higher levels of MVPA.