This work explores the effect of varying punching parameters on the high-cycle fatigue (HCF) response of thick-plate high-strength low-alloy (HSLA) steel intended for heavy-duty truck chassis. A combination of microstructural characterization, tensile testing, HCF experiments on both punched and unpunched specimens, and neutron diffraction-based residual stress measurements was conducted. The punching operation induces pronounced microstructural modifications, including grain refinement, defect generation, tensile residual stresses, development of a hardened shear-affected zone, and a rough fracture surface inside the punched hole. At higher stress amplitudes and shorter fatigue lives (approximately 10⁵ cycles), the HCF behavior after punching remains comparable to that of unpunched specimens and exhibits lower sensitivity to punching-induced changes. In contrast, under lower stress amplitudes and longer lifetimes (around 10⁶ cycles), fatigue strength decreases considerably due to the combined influence of surface roughness and, more critically, tensile residual stresses, with fatigue cracks initiating near mid-thickness—the region of highest measured tensile residual stresses. Furthermore, localized deformation during punching promotes microstructural refinement and defect formation, further influencing fatigue resistance. The optimization of punching parameters to balance the hardening benefits with minimal defect and sub-grain formation can improve fatigue performance. These insights offer strategies to enhance fatigue performance of HSLA steel in heavy-duty truck chassis components.