Objective: There has been little investigation of non-response bias in web-based health surveys. We hypothesised that non-respondents have a higher prevalence of risk behaviours than respondents. Method: In 2005, random samples of students aged 17–25 years from 12 New Zealand tertiary institutions (n=7130) were invited to complete a web-based health behaviour survey, with three e-mail reminders. Early respondents (before 2nd reminder) were compared with late respondents (after 2nd reminder). Late respondents served as a proxy for non-respondents. Results: 2607 students (37%) responded early, 676 (9%) responded late, and 3847 (54%) did not respond. There were differences between early and late respondents in high school binge drinking (38% vs 47%, p=0.002) and non-compliance with physical activity guidelines (12% vs 18%, p=0.004). Differences in overweight/obesity (26% vs 31%, p=0.058), smoking (18% vs 22%, p=0.091) and non-compliance with dietary guidelines (76% vs 77%, p=0.651) were non-significant but point estimates were in the expected direction. Estimated bias in prevalence of risk behaviours was an absolute difference of 1–4% and a relative difference of 0–21%. Conclusion: Respondents whose participation was hardest to elicit reported more risk behaviour. Assuming non-respondents' behaviour is similar or more extreme than that of late respondents, prevalence will have been substantially underestimated.