Limited evidence is available about (non)-representativeness of participants in health-promoting interventions. The Dutch Healthy Primary School of the Future (HPSF)-study is a school-based study aiming to improve health through altering physical activity and dietary behaviour, that started in 2015 (registered in ClinicalTrials.gov on 14-06-2016, NCT02800616). The study has a response rate of 60%. A comprehensive non-responder analysis was carried out, and responders were compared with schoolchildren from the region and the Netherlands using a cross-sectional design. External sources were consulted to collect non-responder, regional, and national data regarding relevant characteristics including sex, demographics, health, and lifestyle. The Chi-square test, Mann-Whitney U test, or Student's t-test were used to analyse differences. The analyses showed that responders (n = 494) were comparable with non-responders (n = 348) and regional data (n = 6172) with regard to sex and health. Responders did not significantly differ from regional data with regard to lifestyle. Responders had significantly higher educated parents compared to non-responders and were more often of autochthonous ethnicity compared to regional data. Major differences were observed between responders and schoolchildren in the Netherlands, regarding, among others sex, ethnicity, and parental employment rates. We conclude that a potential healthy-volunteer effect in the HPSF-sample is limited. External validity is high when compared to the regional population but low when compared to the national sample. For future intervention studies, we advise to evaluate outcome measures according to regional/national standards and to cooperate with external parties in early stages of research to be able to assess and enhance generalisability.