Abstract: The objective of this study was to determine the influence of thermal processing on the assessment of tocopherols and carotenoids, as well as their isomer formation in tomatoes. The sliced tomatoes were heated in an oven at 100, 130, and 160 °C for 5, 10, and 20 min, then freeze-dried. Freeze-dried samples were finely ground and the analysis was performed on lyophilized samples. The average concentrations of total lycopene, lutein, β-carotene, α-tocopherol, and γ-tocopherol in fresh tomatoes (in 100 g dry weight) were 21.2, 1.1, 2.7, 8.0, and 2.5 mg, respectively. Oven baking of tomato at 160 °C for 20 min led to a significant increase in the apparent measurement of lycopene, β-carotene, and α-tocopherol content by 75%, 81%, and 32%, respectively. Heating induced isomerization of (all-E) to various (Z) isomers of lycopene, and we found that the total (Z)-lycopene proportion in the tomatoes increased with longer heating time. (All-E)-lycopene constituted 75.4% in fresh tomatoes and decreased to 52.5% in oven-baked tomatoes (160 °C, 20 min), while (5Z)-lycopene increased from 9.4% to 17.9% of total lycopene. However, β-carotene release and isomerization was less influenced by the heat treatment than that of lycopene. These results suggested that thermal processes might break down cell walls and enhance the release of carotenoids and tocopherols from the matrix, as well as increase isomerization of lycopene and β-carotene.