The aim of the present study was to assess 24-h free IGF-I profiles in serum in healthy children using an ultrafiltration method that approached in vivo conditions. Five girls and two boys aged 10.4 to 13.6 (mean 12.2) years with pubertal stages I to III were studied. A fasting blood sample was drawn at 0800 h, and thereafter samples were drawn at specific times every 20 min until 0800 h the next morning. Free IGF-I, IGF binding protein 1 (IGFBP-1), and insulin were analyzed in 1-h samples, total IGF-1 in 2-h samples, and GH in 20-min samples. A statistically significant diurnal variation in serum free IGF-I was seen (p < 0.001) with peak values between 0900 and 1200 h in the morning and a nocturnal decrease with a nadir at 0700 h (p < 0.05). Concomitantly with the decrease in free IGF-I an increase in IGFBP-1 was observed between 0200 and 0700 h (p < 0.001). Total IGF-I did not exhibit any diurnal variation. Inverse relationships between the 24-h area under the curve (24-hAUC) free IGF-I and 24-hAUC IGFBP-1 (p = 0.002) and between fasting free IGF-I and fasting IGFBP-1 levels (p-0.01) were observed. Furthermore, 24-hAUC GH correlated with fasting free IGF-I (p = 0.04), 24-hAUC free IGF-I (p = 0.03), fasting total IGF-I (p = 0.04), and 24-hAUC total IGF-I (p = 0.04). No phase relationship between free IGF-I and IGFBP-1 or insulin were seen. In healthy children, circulating free IGF-I exhibits a nocturnal decrease and an increase in the morning. The diurnal secretion of free IGF-I correlates with GH and is inversely related to IGFBP-1. The metabolic significance of these findings needs further study.