The reliability of ACF (Anisotropic conductive film) interconnection is a serious concern especially under thermal loading condition. This paper focuses on the online contact resistance behavior of the ACF joint for bumpless flip-chip on flex packages during different thermal cycling conditions. In this work, flip chips of 11×3 mm2 having bare aluminum pad were used. Real time contact resistance (i.e. live measurement contact resistance variation with temperature) was measured by four points probe method when the packages were inside thermal shock chamber. Tests for three different thermal cycling profiles (125°C to −55°C, 140°C to −40°C and 150°C to −65°C) were carried out. The samples bonded at temperature 180°C and pressure of 2.42Mpa was used. The initial contact resistance of the bumpless samples was 0.4Ω. Contact resistance increased with the number of thermal cycles, however the effect was severe when the temperature variation was above the glass transition temperature (Tg) of the ACF matrix (131°C). Differences in co-efficient of thermal expansion (CTE) between the chip and the substrate generated thermal stresses during temperature fluctuation, which caused the pad of the substrate to slide over the Al pad of the chip. Thus variation of the contact resistance was also observed along the interconnection position in the package, i.e. corner joint showed higher value of increase in contact resistance than the middle position. Even though flex substrate was used in this study; the sliding effect was severe at the corner Al pads of the chip, where cumulative forces generated due to the thermal stress. Results show that for thermal cycling profile 140°C to −40°C, online contact resistance increased to 1.2 Ω in corner joint, whereas for the middle joints the contact resistance just increased to 0.5 Ω. Glass transition temperature (Tg) of the ACF material plays an important role on the high temperature contact resistance. For every thermal cycling profile, there is an incubation period that would have significant impact in the application of ACF. After the incubation period the contact resistance increases rapidly and the joints are no longer reliable.