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Objectives: Lyme serology does not readily discriminate an active Lyme borreliosis (LB) from a previous Borrelia infection or exposure. Here, we aimed to investigate a large number of immunological protein biomarkers to search for an immunological pattern typical for active LB, in contrast to patterns found in healthy blood donors, a proportion of whom were previously exposed to Borrelia., Methods: Serum samples from well-characterised adult patients with ongoing LB and healthy blood donors were included and investigated using a proximity extension assay (provided by Olink®) by which 92 different immune response-related human protein biomarkers were analysed simultaneously., Results: In total, 52 LB patients and 75 healthy blood donors were included. The blood donors represented both previously Borrelia exposed (n = 34) and not exposed (n = 41) based on anti-Borrelia antibody status. Ten of the examined 92 proteins differed between patients and blood donors and were chosen for further logistic regression (p<0.1). Six proteins were statistically significantly different between LB patients and blood donors (p<0.05). These six proteins were then combined in an index and analysed using receiver-operating-characteristic curve analysis showing an area under the curve of 0.964 (p<0.001)., Conclusions: The results from this study suggest that there is an immunological protein pattern that can distinguish a present Borrelia infection from a previous exposure as well as anti-Borrelia antibody negative blood donors. Although this method is not adapted for routine clinical use at this point, the possibility is interesting and may open new diagnostic opportunities improving the laboratory diagnostics of LB., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: IT reports participation in advisory board and personal fees from Pfizer Inc outside the submitted work. AJH has a collaborative research agreement with Abbott Laboratories, Chicago, USA and Reagena Oy, Toivala, Finland. Remaining authors report no conflict of interest., (Copyright: © 2023 Tjernberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)