Abstract Background A primary aim of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study is to examine the association between total physical activity levels (comprising occupational, household and recreational activity) and the incidence of cancer. We examined the validity and long-term repeatability of total physical activity measurements estimated from the past-year recall EPIC questionnaire, using accelerometers as an objective reference measure. Methods Participants included 100 men and 82 women aged 50–65 years. Criterion validity was assessed by comparing the physical activity estimates from the EPIC questionnaire with total activity estimated from the average of three separate 7-day accelerometer periods during the same (past-year) period. Long-term repeatability of the EPIC questionnaire was assessed by comparing the responses from the baseline and 10-month administrations. Past-year EPIC estimates were also compared with the Friedenreich Lifetime Total Physical Activity Questionnaire to examine whether recent activity reflected lifetime activity. Results Accelerometer total metabolic equivalent (MET)-hours/week were positively associated with a total physical activity index (Spearman rank correlation ρ = 0.29, 95% confidence interval (CI) 0.15, 0.42) and with non-occupational activity estimated in MET-hours/week (ρ = 0.21, 95% CI 0.07, 0.35). Stratified analyses suggested stronger correlations for non-occupational activity for participants who were male, had a lower BMI, were younger, or were not full-time workers, although the differences in correlations between groups were not statistically significant. The weighted kappa coefficient for repeatability of the total physical activity index was 0.62 (95% CI 0.53, 0.71). Spearman correlations for repeatability of components of activity were 0.65 (95% CI 0.55, 0.72) for total non-occupational, 0.58 (95% CI 0.48, 0.67) for recreational and 0.73 (95% CI 0.66, 0.79) for household activity. When past-year activity was compared to lifetime estimates of activity, there was fair agreement for non-occupational (ρ = 0.26) activity, which was greater for household activity (ρ = 0.46) than for recreational activity (ρ = 0.21). Conclusion Our findings suggest that the EPIC questionnaire has acceptable measurement characteristics for ranking participants according to their level of total physical activity. The questionnaire should be able to identify the presence or absence of reasonably strong aetiological associations when either recent or long-term activity is the .