Identifying in vivo the processes that determine lesion severity in multiple sclerosis (MS) remains a challenge. To describe the dynamics of ultrasmall superparamagnetic iron oxide (USPIO) enhancement in MS lesions and the relationship between USPIO enhancement and microstructural changes over 3 years. Lesion development was assessed at baseline, Months 3, 6, and 9, using magnetic resonance imaging (MRI) with gadolinium and USPIO. Microstructural changes were assessed at baseline, Months 3, 6, 9, 12, 18, 24, and 36, using relaxometry, magnetization transfer, and diffusion-weighted imaging. We included 15 patients with clinically isolated syndrome. In the 52 MRI scans acquired with USPIO, 22 lesions were USPIO and gadolinium positive, and 44 were USPIO negative but gadolinium positive. Lesions no longer exhibited sustained USPIO enhancement 3 months later. At baseline, lesions that were both USPIO and gadolinium positive had lower magnetization transfer ratio values (common language effect size = 0.84, p = 0.0005) and lower fractional anisotropy values (0.83, p = 0.001) than gadolinium-positive-only lesions. USPIO-positive lesions remained associated with greater damage than gadolinium-positive-only lesions throughout the 3-year follow-up. USPIO enhancement, mainly reflecting monocyte infiltration, is transient and is associated with persistent tissue damage after 3 years.