Background <p>With increasing ultrasound use, data for the normal amount of joint fluid and synovial appearances are needed.</p> Objective <p>To establish reference values for knee-ultrasound in healthy volunteers by age. To examine the association of joint fluid and synovial appearances with age, sex, and time spent in sports activities.</p> Materials and methods <p>Prospective, cross-sectional, including 3–17-year-old volunteers. Examinations were performed by one of two experienced radiologists after meticulous standardisation of the technique. Knees were examined in 30° flexion, in extension and during provocation.</p> Results <p>One hundred twenty-seven volunteers (67 females), median age 10.9&#xa0;years (percentile 2.5^th-97.5^th: 3.4–17.2), were included. With 30° knee flexion, the median amount of suprapatellar recess joint fluid (sagittal view) was 0.5&#xa0;mm for females and 0.9&#xa0;mm for males (<i>P</i> = 0.014), increasing to 1.9&#xa0;mm and 2.4&#xa0;mm (<i>P</i> = 0.031), respectively, during provocation. The amount of fluid during provocation increased with age for both sexes (<i>P</i> = 0.007 females; <i>P</i> = 0.012 males) and by time spent in sport-activities (<i>P</i> = 0.003). Further, there was a positive association between the presence of joint fluid speckles and sport-activities (<i>P</i> = 0.039). Median double-layered synovium was 1.5&#xa0;mm in 3–6-year-olds, increasing to 2.1&#xa0;mm in 7–9-year-olds and 2.0&#xa0;mm in 10–17-year-olds (<i>P</i> = 0.024). No synovial Doppler signal was seen. Observers’ agreement was excellent, with ICC values ≥ 0.9.</p> Conclusion <p>The majority of healthy children have measurable knee joint fluid, more so when examined with a flexed knee and under provocation. Speckles within the joint fluid are seen in one-fourth, especially in males, and the presence of speckles and joint fluid amount are associated with sports-activity.</p> Graphical Abstract <p></p>