Background: Ovarian cancer is a high mortality gynecological malignancy with poor survival rates, where patients might benefit from early stage detection strategies. The United Kingdom Collaborative Trial for Ovarian Cancer Screening (UKCTOCS) study employed a multi-modal screening (MMS) strategy utilizing longitudinal CA125 values interpreted with a risk of ovarian cancer algorithm (ROCA) as a first line screen to triage women for confirmatory transvaginal ultrasound. MMS achieved a sensitivity of 84% for invasive epithelial ovarian/tubal cancer (iEOC) diagnosed within 1 year of screens. Despite the promise of this strategy, about 15-20% of iEOC do not express CA125 and will be missed. We previously identified a panel of biomarkers comprising CA125, HE4, MMP-7 and CA 72-4 that could distinguish early stage ovarian cancers from healthy individuals with 83% sensitivity at 98% specificity. Methods: In this work, we retrospectively assessed pre-clinical longitudinal specimens from 75 women destined to develop ovarian cancer (3-5 serial specimens each) and 525 corresponding controls (5 serial specimens each) from the UKCTOCS study, in a blinded trial, to estimate complementarity and lead times for the biomarker panel in comparison to CA125. Of the 75 cases, 50 were CA125 screen positive and 25 were CA125 screen negative. We developed flow cytometric bead-based immunoassays for the biomarker panel with low %CVs and limits of detection suitable for assessment of low biomarker concentrations to permit assessment using ultra-low (16 µL) volumes of serum. Results: One or more markers rose with CA125 in the screen positive samples, however, no lead times were observed. We developed single marker longitudinal algorithms to determine the presence of a change point to distinguish between the cases and controls, similar in principle to the ROCA algorithm. Based on this algorithm, HE4 and CA72-4 identified 16% of cases missed by CA125 at 98% specificity. MMP-7 did not identify any cases missed by CA125. Conclusions: HE4 and CA72-4 show promise to complement CA125 in a longitudinal screening strategy.