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β-alanine supplementation increases muscle carnosine content and improves anaerobic exercise performance by enhancing intracellular buffering capacity. β-alanine ingestion in its traditional rapid-release formulation (RR) is associated with the symptoms of paresthesia. A sustained-release formulation (SR) of β-alanine has been shown to circumvent paresthesia and extend the period of supply to muscle for carnosine synthesis. The purpose of this investigation was to compare 28 days of SR and RR formulations of β-alanine (6 g day-1) on changes in carnosine content of the vastus lateralis and muscle fatigue. Thirty-nine recreationally active men and women were assigned to one of the three groups: SR, RR, or placebo (PLA). Participants supplementing with SR and RR formulations increased muscle carnosine content by 50.1% (3.87 mmol kg-1ww) and 37.9% (2.62 mmol kg-1ww), respectively. The change in muscle carnosine content in participants consuming SR was significantly different (p = 0.010) from those consuming PLA, but no significant difference was noted between RR and PLA (p = 0.077). Although participants ingesting SR experienced a 16.4% greater increase in muscle carnosine than RR, fatigue during maximal voluntary isometric contractions was significantly attenuated in both SR and RR compared to PLA (p = 0.002 and 0.024, respectively). Symptoms of paresthesia were significantly more frequent in RR compared to SR, the latter of which did not differ from PLA. Results of this study demonstrated that only participants consuming the SR formulation experienced a significant increase in muscle carnosine. Differences in the muscle carnosine response between these formulations may have practical significance for athletic populations in which small changes may have important implications on performance.