BACKGROUND: Ischemic stroke is among the most prevalent diseases, with high death and morbidity. Numerous preclinical studies have reported efficacious interventions in rodent stroke models. However, reperfusion therapies remain the only clinically efficacious intervention to date. Rigor and reproducibility are now recognized as critical to bridge the preclinical-clinical disconnect. Inter-α inhibitor proteins (IαIPs) are a family of structurally related glycoproteins with 2 major forms (inter-α inhibitor and pre-α inhibitor) in blood. Purified human plasma-derived IαIP has beneficial effects in sepsis and hypoxic-ischemic brain injury. More recently, IαIP improved focal ischemic stroke outcomes in mouse models. Here, we tested IαIP efficacy in both transient and permanent stroke mouse models, mimicking previously published study designs and protocols to seek reproducibility. METHODS AND RESULTS: Using healthy young male and female C57BL/6 mice, we induced transient or permanent endovascular filament middle cerebral artery occlusion (MCAO). Mice were divided into transient MCAO+vehicle, transient MCAO+IαIP (30 mg/kg), permanent MCAO+vehicle, and permanent MCAO+IαIP groups. IαIP or vehicle was administered intravenously at 6 and 18 hours after MCAO. End points were assessed at 2 days. Efficacy readouts included death, infarct volume and swelling, and 3 neurological tests. Contrary to the previous work, we did not find IαIP efficacious on any outcome readout in either transient MCAO or permanent MCAO. CONCLUSIONS: Our data highlight the contribution of interlaboratory heterogeneity to study outcomes and suggest that interventions considered for clinical development should undergo rigorous testing in multiple single-laboratory studies before entering a multicenter preclinical trial.