Some marine microalgae, such as Isochrysis sp., produce high-melting (∼70 °C) lipids known as long-chain alkenones that detrimentally affect biodiesel fuel quality. A method has been developed for the production of an alkenone-free Isochrysis biodiesel. This material was prepared on sufficient scale to allow for extensive analysis according to ASTM standards. Results revealed that while cold flow improved by removal of these high-melting components and lubricity and glycerol content were below maximum levels prescribed in the standards, other properties dependent on fatty acid methyl ester (FAME) structure and composition such as oxidative stability failed to meet ASTM specifications. Both the cetane number and kinematic viscosity were lower than what would be calculated based on FAME content. This has been explained in part by 6–8% non-FAME contaminants in the tested samples.