We assessed the reproducibility of measurements of plasma hormone and binding protein levels in umbilical cord blood collected from 30 male and female babies. They were delivered as singleton births from full-term pregnancies (gestational age ≥ 37 weeks) in a cord blood donation programme. We assayed three plasma replicates from each cord blood sample at two points in time. Plasma oestradiol, unconjugated oestriol, testosterone, progesterone, prolactin, sex-hormone binding globulin (SHBG), insulin-like growth factor-1 (IGF-1), and IGF binding protein-3 (IGFBP-3) levels were measured in duplicates in the same batch (batch 1). In addition, another set of assays was conducted for each cord blood 1 year apart in a different batch (batch 2). Means and standard deviations for each hormone and binding protein were similar in replicates assayed in batch 1 and 2. Pearson's correlation coefficients were 0.9 or higher in duplicates assayed in batch 1. The correlation coefficients were between 0.77 and 0.96 for between-batch assays. Intra-class correlation coefficients (ICC) were higher than 0.9 for assay of SHBG [95% CI 0.92, 1.0] and progesterone [95% CI 0.87, 0.97] and between 0.8 and 0.9 for assay of oestradiol, unconjugated oestriol, prolactin, IGF-1, and IGFBP-3. The lowest ICC value was found for testosterone (ICC = 0.74; [95% CI 0.56, 1.0]). These data indicate a high reproducibility of cord blood hormone measurements; minimal differences were observed between the calibrated and the original regression coefficients for the association of hormones/binding proteins with percent of CD34+ cells in mononuclear cells.