Abstract Background T-wave inversion (TWI) is rare in athlete's heart but is a common manifestation in cardiomyopathies. Although TWI has been extensively investigated in adult athletes, the ability of this ECG pattern to distinguish between a physiological variant and a developing heart muscle disease in children is controversial. The aim of this longitudinal study was to establish the prevalence, changes and clinical significance of TWI in a large cohort of pre-adolescent athletes. Methods 2227 children (mean age 12.3 ± 2.0 years) undergoing sports preparticipation screening were included. Children with TWI underwent yearly follow-up until the positivisation of TWI for a maximum follow-up of 4 years. Results Among 2227 children, 358 (16%) had TWI. Children with TWI were younger (11.4 ± 2.1 vs. 12.5 ± 2.0 years, p < 0.0001) and had a lower BSA than children without TWI (p < 0.0001). 97% of children showed anterior TWI while only 3% had infero-lateral TWI. Anterior TWI became positive in 94% of children during the 4-year follow-up (p < 0.0001 vs. baseline) and the remaining 6% did not show abnormal clinical findings. Conversely, in the group of 9 children with infero-lateral TWI, only 1 showed normalisation during follow-up (p = 0.81) and 1 was found to have a cardiomyopathy. Conclusions Anterior TWI is common in children and generally becomes positive by the age of 14 years. Conversely, infero-lateral TWI is rare, persistent and may be associated with structural heart disease. Therefore, infero-lateral TWI should not be interpreted as physiologically related to age, development or training and children with infero-lateral TWI should remain under strict clinical surveillance. Highlights • This study establishes the prevalence of TWI in a large cohort of children practising sport • This longitudinal study demonstrates that anterior TWI usually becomes positive by the age of 14 years • Conversely, children with infero-lateral TWI followed for 4 years did not demonstrate significant changes in the ECG pattern • Infero-lateral TWI should not be interpreted as physiologically related to age, development or training • This study support the concept that children with infero-lateral TWI should remain under strict clinical surveillance [ABSTRACT FROM AUTHOR]