BACKGROUND: Accelerometers are widely used to measure sedentary time and daily physical activity (PA). However, data collection and processing criteria, such as non-wear time rules might affect the assessment of total PA and sedentary time and the associations with health variables. The study aimed to investigate whether the choice of different non-wear time definitions would affect the outcomes of PA levels in youth. METHODS: Seventy-seven healthy youngsters (44 boys), aged 10-17 years, wore an accelerometer and kept a non-wear log diary during 4 consecutives days. We compared 7 published algorithms (10, 15, 20, 30, 60 min of continuous zeros, Choi, and Troiano algorithms). Agreements of each algorithm with the log diary method were assessed using Bland-Altmans plots and by calculating the concordance correlation coefficient for repeated measures. RESULTS: Variations in time spent in sedentary and moderate to vigorous PA (MVPA) were 30 and 3.7%. Compared with the log diary method, greater discrepancies were found for the algorithm 10 min (p < 0.001). For the time assessed in sedentary, the agreement with diary was excellent for the 4 algorithms (Choi, r = 0.79; Troiano, r = 0.81; 30 min, r = 0.79; 60 min, r = 0.81). Concordance for each method was excellent for the assessment of time spent in MVPA (> 0.86). The agreement for the wear time assessment was excellent for 5 algorithms (Choi r = 0.79; Troiano r = 0.79; 20 min r = 0.77; 30 min r = 0.80; 60 min r = 0.80). CONCLUSIONS: The choice of non-wear time rules may considerably affect the sedentary time assessment in youth. Using of appropriate data reduction decision in youth is needed to limit differences in associations between health outcomes and sedentary behaviors and may improve comparability for future studies. Based on our results, we recommend the use of the algorithm of 30 min of continuous zeros for defining non-wear time to improve the accuracy in assessing PA levels in youth. TRIAL REGISTRATION: NCT02844101 (retrospectively registered at July 13th 2016).