Abstract Background Physical activity (PA) has acquired a significant relevance due to the health benefits associated with its practice. Accelerometers are an effective tool to assess PA; however, the diversity of cut-off points used to define different PA intensities through accelerometry could interfere in the interpretation of the findings among studies. Objectives The present study aimed to examine the sedentary behavior (SB) and physical activity (PA) levels in children using six selected accelerometry protocols based on diverse cut-off points. Methods Clinical examination, anthropometric measurements, and PA evaluation by accelerometry were assessed in 543 selected children (10 ± 2.4 years old) from the Spanish GENOBOX study. The ActiLife data scoring program was used to determine daily min spent in SB, and light, moderate, vigorous and moderate-vigorous PA using six validated accelerometry protocols differing in their cut-off points. Results Very different estimations for SB and PA intensity levels were found in children, independently of the non-wear-time algorithm selected, and considering puberty stages, age and body mass index. The time spent in daily SB varied from 471 to 663.7 min, PA ranged from 141 to 301.6 min, and the moderate-vigorous PA was reported between 20.7 and 180.2 min. Conclusion The choice of a particular accelerometry protocol considering these factors is important to evaluate SB or PA intensities to suit the characteristics of the sample researched. It seems necessary to establish future lines of research that include different analytical approaches to measure SB and PA by accelerometry based on standardized and validated methodology.