Background: Pediatric differentiated thyroid cancer (DTC) has an excellent prognosis, but unknown late effects of treatment. Initial cardiac evaluation showed subclinical diastolic dysfunction in 20% of adult survivors. The objective of this follow-up study was to determine the clinical course of this finding. Methods: This multicenter study, conducted between 2018 and 2020, re-evaluated survivors after 5 years. The primary endpoint was echocardiographic diastolic cardiac function (depicted by the mean of the early diastolic septal and early diastolic lateral tissue velocity [e’ mean]). Secondary endpoints were other echocardiographic parameters and plasma biomarkers. Results: Follow-up evaluation was completed in 47 (71.2%) of 66 survivors who had completed their initial evaluation. Of these 47 survivors, 87.2% were women. Median age was 39.8 years (range 18.8-60.3), and the median follow-up after initial diagnosis was 23.4 years (range 10.2-48.8). Between the first and second evaluation, the e’ mean significantly decreased by 2.1 cm/s (SD 2.3 cm/s, P<0.001). The median left ventricular ejection fraction did not significantly change (58.0% vs. 59.0%, NS). In the best explanatory model of e’ mean, multivariate linear regression analysis showed that body mass index and age were significantly associated with e’ mean (β coefficient -0.169 , 95% confidence interval (CI) [-0.292;-0.047], P=0.008 and β coefficient -0.177 ,95% CI [-0.240;-0.113], P<0.001, respectively). Conclusions and relevance: In these relatively young survivors of pediatric DTC, diastolic function decreased significantly during 5 year follow up and is possibly more pronounced than in normal ageing. This finding requires further follow-up to assess clinical consequences.