The association between treatment-related lymphopenia in multiple sclerosis, drug efficacy and the risk of infections is not yet fully understood. The objective of this study was to assess whether lymphopenia is associated with short-term treatment response and infection rate in a real-life multiple sclerosis population treated with fingolimod and dimethyl-fumarate. We assessed the associations between baseline absolute lymphocyte count and the lymphocyte mean percentage decrease at 6 and 12months with treatment response and the occurrence of adverse events over 12months in the entire cohort of patients and in the two treatment groups separately. This is a retrospective observational real-world study of patients with multiple sclerosis treated with fingolimod and dimethyl-fumarate at the MS Center of the University of Genoa between 2011 and 2018. Patients with at least 12months of follow-up were eligible if [1] they had an Expanded Disability Status Scale assessment at baseline and 12months after treatment onset, [2] they had undergone brain magnetic resonance imaging at baseline and after 12months, and [3] absolute lymphocyte counts were available at baseline, 6 and 12months. Patients shifting from dimethyl-fumarate to fingolimod or vice versa were excluded from the analysis. In total, 137 and 75 patients treated with fingolimod and dimethyl-fumarate, respectively, were included in the analysis. At 12 months,fingolimod-treated patientswere more likely to experience grade II and grade IIIlymphopenia compared with dimethyl-fumarate patients (p<0.001,χ2=94) and hada higher lymphocytemean percentage decrease (p < 0.001, U = 540). A higher number of previous therapies and a lower baseline absolute lymphocyte count were predictors of lymphopenia at 6months (p = 0.047, odds ratio = 1.60 and p = 0.014, odds ratio = 1.1) and 12months (p = 0.003, odds ratio = 1.97 and p = 0.023, odds ratio = 1.1). In fingolimod-treated patients only, female sex and a higher Expanded Disability Status Scale score were predictors of lymphopenia at 12months (p = 0.006, odds ratio = 7.58 and p = 0.03, odds ratio = 1.56). Neither absolute lymphocyte count at 6 and 12 months nor the mean percentage decrease at 6 and 12 months predicted No Evidence of Disease Activity (NEDA-3) status at 1 year, the occurrence of relapses, disease activity on MRI or disability progression. Our findings suggest that peripheral blood lymphocyte changes are not associated with short-term treatment response and with the rate of infections during fingolimod and dimethyl-fumarate treatment in real-world patients. Higher treatment exposure and a lower baseline absolute lymphocyte count are risk factors for lymphopenia development during fingolimod and dimethyl-fumarate therapy.