Background: Early differentiation between transient ischemic attack (TIA) and minor ischemic stroke (MIS) impacts on the patient's individual diagnostic work-up and treatment. Furthermore, estimations regarding persisting impairments after MIS are essential to guide rehabilitation programs. This study evaluated a combined clinical- and serum biomarker-based approach for the differentiation between TIA and MIS as well as the mid-term prognostication of the functional outcome, which is applicable within the first 24 h after symptom onset.Methods: Prospectively collected data were used for a retrospective analysis including the neurological deficit at admission (National Institutes of Health Stroke Scale, NIHSS) and the following serum biomarkers covering different pathophysiological aspects of stroke: Coagulation (fibrinogen, antithrombin), inflammation (C reactive protein), neuronal damage in the cellular [neuron specific enolase], and the extracellular compartment [matrix metalloproteinase-9, hyaluronic acid]. Further, cerebral magnetic resonance imaging was performed at baseline and day 7, while functional outcome was evaluated with the modified Rankin Scale (mRS) after 3, 6, and 12 months.Results: Based on data from 96 patients (age 64 ± 14 years), 23 TIA patients (NIHSS 0.6 ± 1.1) were compared with 73 MIS patients (NIHSS 2.4 ± 2.0). In a binary logistic regression analysis, the combination of NIHSS and serum biomarkers differentiated MIS from TIA with a sensitivity of 91.8% and a specificity of 60.9% [area under the curve (AUC) 0.84]. In patients with NIHSS 0 at admission, this panel resulted in a still acceptable sensitivity of 81.3% (specificity 71.4%, AUC 0.69) for the differentiation between MIS (n = 16) and TIA (n = 14). By adding age, remarkable sensitivities of 98.4, 100, and 98.2% for the prediction of an excellent outcome (mRS 0 or 1) were achieved with respect to time points investigated within the 1-year follow-up. However, the specificity was moderate and decreased over time (83.3, 70, 58.3%; AUC 0.96, 0.92, 0.91).Conclusion: This pilot study provides evidence that the NIHSS combined with selected serum biomarkers covering pathophysiological aspects of stroke may represent a useful tool to differentiate between MIS and TIA within 24 h after symptom onset. Further, this approach may accurately predict the mid-term outcome in minor stroke patients, which might help to allocate rehabilitative resources.