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The purpose of this study was to analyse differences between total physical activity (TPA) and moderate-to-vigorous PA (MVPA) of pre-school children during daily school hours when they attended the physical education class (PED) and school days without PE class (NPED) and to assess the contribution of PE classes to TPA in school hours. The sample was composed of 193 pre-school healthy children (96 girls) aged from three to five years old and was conducted between February and December of 2008. Children wore accelerometers for at least four consecutive days during school hours. Data were analysed with specific software, age-specific counts-per-minute cut-off points and a 5 s epoch were used. Independent and general linear model repeated measures were used to assess differences between gender and differences between different days within each gender, respectively. Boys engaged more MVPA than girls (P < 0.05). During PED, pre-school children engaged significantly more in TPA and MVPA than during NPED (P < 0.05). PE class contributed, on average, 27.7% for the TPA and 32.8% of daily MVPA during PED in both gender. The results of this study suggest that structured PA such as a PE class increased the daily TPA and MVPA level of pre-school children.