Macamides are a class of amide alkaloids that are only found in maca and are widely considered to be its bioactive marker compounds. More than thirty macamide monomers have been identified in recent years; however, it is difficult to obtain a single macamide monomer from the maca plant because of their similar structures and characteristics. We used the carbodiimide condensation method (CCM) to efficiently synthesize five typical macamides, including N-benzyl-hexadecanamide (NBH), N-benzyl-9Z,12Z,15Z-octadecenamide, N-(3-methoxybenzyl)-9Z,12Z-octadecenamide, N-benzyl-9Z,12Z-octadecenamide, and N-(3-methoxybenzyl)-9Z,12Z,15Z-octadecadienamide. All the synthesized macamides were purified by a one-step HPLC with a purity of more than 95%. NBH is the most abundant macamide monomer in natural maca, and it was selected to evaluate the anti-fatigue effects of macamides. The results indicated that NBH could enhance the endurance capacity of mice by increasing liver glycogen levels and decreasing blood urea nitrogen, lactate dehydrogenase, blood ammonia, and blood lactic acid levels. Macamides might be the active substances that give maca its anti-fatigue active function.