A D-optimal mixture design was used to study the effects of onion and extra virgin olive oil (EVOO) on lycopene Z-isomerization, lycopene diffusion into oil (expressed as a partition factor between tomato-based puree and oil) and in vitro bioaccessibility of lycopene isomers after thermal treatment of tomato-based puree consisting of tomato (75-100%), onion (0-20%) and EVOO (0-5%). A decrease of tomato puree could improve lycopene Z-isomerization, lycopene diffusion and lycopene bioaccessibility. The component interactions had an important influence on the Z-isomerization of lycopene, besides the linear mixtures of components. However, only linear mixtures of components appeared to have significant effects on the diffusion and bioaccessibility of lycopene, in which EVOO had the highest positive effect followed by onion. The bioaccessibility of lycopene isomers in every tomato-based sauce formulation decreased in the order: 13-Z-lycopene > 9-Z-lycopene > 5-Z-lycopene > all-E-lycopene. The bioaccessibility of total-Z-lycopene was at least 10 times higher than that of all-E-lycopene. Proportions of total-Z-lycopene were correlated positively with the partition factor and bioaccessibility of total-lycopene, with an r over 0.730 (p = 0.0031). Therefore, increased Z-lycopene proportions probably contributed to enhanced lycopene diffusion and bioaccessibility. The positive effects of components, especially onion, on total-lycopene diffusion and bioaccessibility were probably because the components increased the Z-isomerization of lycopene during heating of tomato-based puree.