Background: Training in the five steps of evidence-based practice (EBP) has been recommended for inclusion in entry-level health professional training. The effectiveness of EBP education has been explored predominantly in the medical and nursing professions and more commonly in post-graduate than entry-level students. Few studies have investigated longitudinal changes in EBP attitudes and behaviours. This study aimed to assess the changes in EBP knowledge, attitudes and behaviours in entry-level physiotherapy students transitioning into the workforce. Methods. A prospective, observational, longitudinal design was used, with two cohorts. From 2008, 29 participants were tested in their final year in a physiotherapy program, and after the first and second workforce years. From 2009, 76 participants were tested in their final entry-level and first workforce years. Participants completed an Evidence-Based Practice Profile questionnaire (EBP 2), which includes self-report EBP domains [Relevance, Terminology (knowledge of EBP concepts), Confidence, Practice (EBP implementation), Sympathy (disposition towards EBP)]. Mixed model analysis with sequential Bonferroni adjustment was used to analyse the matched data. Effect sizes (ES) (95% CI) were calculated for all changes. Results: Effect sizes of the changes in EBP domains were small (ES range 0.02 to 0.42). While most changes were not significant there was a consistent pattern of decline in scores for Relevance in the first workforce year (ES -0.42 to -0.29) followed by an improvement in the second year (ES +0.27). Scores in Terminology improved (ES +0.19 to +0.26) in each of the first two workforce years, while Practice scores declined (ES -0.23 to -0.19) in the first year and improved minimally in the second year (ES +0.04). Confidence scores improved during the second workforce year (ES +0.27). Scores for Sympathy showed little change. Conclusions: During the first two years in the workforce, there was a transitory decline in the self-reported practice and sense of relevance of EBP, despite increases in confidence and knowledge. The pattern of progression of EBP skills beyond these early professional working years is unknown.