Regular physical activity (PA) during childhood is associated with a range of positive health outcomes and higher educational attainment. However, only 2.0% to 14.7% of girls and 9.5% to 34.1% of boys are meeting the current PA guidelines of 60 minutes of moderate to vigorous PA daily. Schools are targeted as a key setting to improve children's PA levels. The Daily Mile (TDM), a teacher-led 15 minute PA intervention was established in 2012 and has been widely adopted globally. However, the dynamic school environment generates challenges for school-based interventions to follow a uniform implementation method resulting in sustainability issues and limited evaluation. The aims of this mixed-methods study were to (1) explore whether whole-school experiences of TDM were related to implementation and (2) examine the association between TDM and CRF in children from high and low socio-economic groups. Focus groups with pupils (n = 6) and interviews with teachers (n = 9) and headteachers (n = 2) were conducted to explore factors associated with successful implementation. Pupils (n = 258 imputed) aged 9-11 from six primary schools in south Wales, United Kingdom participated in CRF assessments (20m shuttle run test) at two time-points (baseline, 6 month follow-up). Thematic analyses of qualitative measures and linear regression analyses of quantitative measures were used to assess the research questions. Qualitative findings identified implementation factors associated with a positive experience of TDM; flexible and adaptable, not replacing current play provision but delivered as an additional playtime, incorporate personal goal setting, teacher participation, whole-school delivery with community support. Both groups demonstrated equal increases in shuttles between baseline and follow-up (deprived: 4.7 ± 13.4, non-deprived: 4.8 ± 16.0). There was no significant difference in this increase for deprived compared to non-deprived children adjusted for age and gender. Findings from this study provide a set of recommendations for the future implementation and sustainability of TDM.