Muscle carnitine palmitoyltransferase II (CPT II) deficiency is associated with various mutations in CPT2 gene. In the present study, the impact of the two CPT II variants P50H and Y479F were characterized in terms of stability and activity in vitro in comparison to wildtype (WT) and the well investigated variant S113L. While the initial enzyme activity of all variants showed wild-type-like behavior, the activity half-lives of the variants at different temperatures were severely reduced. This finding was validated by the investigation of thermostability of the enzymes using nano differential scanning fluorimetry (nanoDSF). Further, it was studied whether the protein stabilizing diphosphatidylglycerol cardiolipin (CL) has an effect on the variants. CL indeed had a positive effect on the stability. This effect was strongest for WT and least pronounced for variant P50H. Additionally, CL improved the catalytic efficiency for CPT II WT and the investigated variants by twofold when carnitine was the varied substrate due to a decrease in KM. However, there was no influence detected for the variation of substrate palmitoyl-CoA. The functional consequences of the stabilization by CL in vivo remain open.