Abstract: In-phase (IP) and out-of-phase (OP) thermal-mechanical fatigue (TMF) tests of 4Cr5MoSiV1 hot work die steel were conducted in full reverse mechanical strain control in the temperature range of 400-700℃ by a TMF servo-hydraulic testing system (MTS^®). The results indicate that, when the strain amplitude is ±0.50%, the OP TMF life of 4Cr5MoSiV1 steel is~60% of the IP TMF life. The stress-strain hysteresis loops show asymmetries for both IP and OP loading. IP loading leads to comprehensive mean stress, while OP loading gives rise to tensile mean stress in the temperature range of 400-700℃. The changes of maximum strain and peak temperature with maximum stress are inconsistent, and the stress relaxation phenomenon could be observed under IP and OP loading. Moreover, two kinds of TMF cycling exhibite continuous cyclic softening in the high temperature half stage, while in the low temperature half stage, cyclic hardening occurs initially and is then followed by continuous cyclic softening. The fractured surfaces under IP TMF loading display striation and tear ridge, and exhibits quasi-cleavage characteristics. In addition, the cracks are less but longer. However, fractured surfaces under OP TMF loading mainly display striation and dimple characteristics, and the cracks are shorter and more abundant.