Background: Infections with Epstein-Barr virus (EBV) and human herpesvirus-6 (HHV-6) have been implicated in multiple sclerosis (MS) onset but little work has studied their relationships in early disease. Objective: Evaluate associations between markers of EBV and HHV-6 infection/reactivation and MS conversion, relapse and EDSS/MSSS amongst 205 CIS participants with EBV/HHV-6 data followed over 5 years. Method: Baseline serological and viral load measures of EBV and HHV-6 exposure/reactivation were measured and infectious mononucleosis (IM) history recorded. Conversion to MS and relapses were assessed annually, and EDSS/MSSS measured at 5-year review. Determinants of MS conversion and relapse assessed by Cox regression, and disability progression by linear regression. Results: IM history showed a strong positive trend with higher relapse risk (aHR=1.45,95%CI=0.97–2.16) but was not associated with MS conversion (aHR=0.92,95%CI=0.57–1.48). Anti-HHV-6 IgG titre>40 also showed strong positive trends with higher relapse (aHR=1.61,95%CI=0.99–2.63) and MS conversion risks (aHR=1.48,95%CI=0.89–2.46). Anti-HHV-6 IgG titre≥640 was significantly associated with higher MSSS (0.15(95%CI=0.00,0.30) and also showed a strong positive trend with higher EDSS 0.10(95%CI=-0.02,0.21). HHV-6 DNA detection showed strong positive trends with 83%(95%CI=-6–357) and 77%(95%CI=-4–328) higher MS conversion and relapse risk. Anti-EBV-EA-D IgG titre was associated with a lower annualised disability progression by EDSS (ptrend=0.037) and also showed strong positive trend with higher MSSS (ptrend=0.053). No associations were seen for other serological or viral load markers. Conclusion: Overall, our data provides evidence that higher HHV-6 IgG was associated with increased risk of MS conversion and relapse but of borderline significance, and greater annualised disability progression, while that for EBV was more limited.