Three pure trehalose fatty acid monoesters (TMs) with different fatty acid substitutions and their mixtureswere assessed for their ability to preserve a lactate dehydrogenase (LDH) activity during freeze/thawprocesses. 6-O-dodecanoyl trehalose (TM12) exhibited a prominent effect for preserving LDH activity.6-O-hexadecanoyl trehalose (TM16) and 6-O-oleoyl trehalose (TM18:1), were less effective, possiblybecause of the tendency for TM16 to precipitation during freeze/thaw cycles and a lower surface tensionreduction ability for TM18:1. TM18:1 was about as effective as TM16. Notably, the gradual replacement ofTM12 with TM18:1 dose-dependently reduced the ability of TMs to preserve LDH activity, and nosignificant mixing effect was observed, whereas replacement with TM16 afforded the positive deviationfrom the expected curve. Inhibition of TM16 precipitation during freeze/thaw processes by TM12 mightfacilitate TM16 performance. The effectiveness of a mixing strategy of the stabilizer was confirmed.