Background: Parkinson’s disease is the second most common neurodegenerative disease worldwide. Accumulating evidence are linking the misfolding and aggregation of the protein alpha-synuclein leading to generation of fibrils, with the disease progression. Fibrillation of alpha-synuclein in vitro can be achieved by induced aggregation of recombinant monomer and termed pre-formed fibrils. When controlling the parameters during fibrillation, different fibril strains can be generated. The strains are hypothesized to induce different diseases due to their different structures. Aim: To obtain different strains of pre-formed fibrils presenting different surface structures and to obtain biophysical characteristics of highly active pre-formed fibrils. Methods: Generation of the multiple forms of pre-formed fibrils were executed by varying chosen physical and chemical parameters. The fibril preparations were analyzed for a number of biophysical and surface structure properties. These includes size distribution, amount of beta-sheet structure formation, zeta potential and limited proteolysis by proteinase K. Furthermore, determination of interaction with a solid support lipid bilayer and measurement of fibril activity were conducted. Results: Variation of physical and chemical parameters during fibrillation generates fibrils of different surfaces assessed by their structural properties. These included differences in degradation patterns in response to proteinase K and behavior in response to a lipid bilayer. Seeding activity of fibrils is dependent on the size distribution. The choice of buffer, concentration and pH value has shown to affect the size of fibrils. Conclusions: Pre-formed fibrils of two strains with different surface structures were generated successfully. While the results indicate different strains are formed, advanced analysis is necessary to evaluate the extent of the differences that are present in the three-dimensional structures of these. We identified key parameters affecting the seeding activity when measured in vitro such as fibril size distribution. Data suggests that these are also affected by buffer composition and protein concentration.