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Seaweed is gaining attention as a possible alternative and sustainable source of proteins. This study investigates three protein extraction methods and their effect on protein yield and quality when applied to Ulva fenestrata. Two of the methods included alkaline extractions (pH-shifts); one version solubilizing the proteins at pH 8.5 and one solubilizing them at pH 8.5 followed by pH 12 (pH 8.5 + 12). The third method was a mechanical pressing, using a double screw press. All extraction methods were followed by isoelectric precipitation to concentrate the proteins. Extraction at pH 8.5 gave the significantly highest total protein yield after the isoelectric precipitation, followed by extraction at pH 8.5 + 12 and lastly mechanical extraction gave the lowest yield. Proteins extracted with both alkaline methods had a significantly higher solubility at pH 7 and pH 9, compared to proteins from the mechanical pressing. There were no significant differences between the three methods in total D/L-amino acid ratio. Amino acid cross-links measured as lysinoalanine (LAL) and lanthionine (LAN) where found in significantly higher amounts in alkali-extracted proteins compared to mechanically extracted, however not to a degree that expect to compromise functional or nutritional quality. Further, no significant difference in protein in vitro digestibility was found between extraction methods. In conclusion, results indicated that protein extraction at pH 8.5 can be recommended, especially regarding total protein yield and solubility of the final protein extract. [Display omitted] • Comparison of mechanical and pH-shift extraction regarding protein yield and protein quality • Alkaline extraction (pH 8.5) was superior to mechanical pressing in protein yield • Alkaline protein extracts had the highest protein solubility in aqueous solution • Alkaline treatment induced cross-linkages depending on the pH • No differences were observed in protein in vitro digestibility between extractions [ABSTRACT FROM AUTHOR]