Purpose A self‐controlled study to determine the influence of illuminance and correlated colour temperature (CCT) of light‐emitting diode (LED) lighting on asthenopia. Methods Twenty‐two healthy postgraduates (nine women) were recruited to read under eight LED lighting conditions with four illuminances (300 lx, 500 lx, 750 lx and 1000 lx) and four CCTs (2700, 4000, 5000 and 6500 K) for 2 h. A subjective asthenopia questionnaire, the optical quality analysis system (OQAS) and an inflammatory cytokine assay were used to assess the levels of asthenopia. Results Increased asthenopia was observed after reading, but the degree varied with lighting conditions. There were significant differences among the groups in terms of subjective symptoms (inattention, eye pain, dry eye and total score), optical performance parameters (modulation transfer function [MTF] cut‐off frequency, Strehl ratio [SR], objective scattering index [OSI], mean OSI and accommodative amplitude [AA]) as well as inflammatory cytokines in the tears (epidermal growth factor [EGF], transforming growth factor [TGF]‐α, interleukin [IL]‐6, IL‐8, macrophage inflammatory protein [MIP]‐1β, tumour necrosis factor [TNF]‐α, TNF‐β and vascular endothelial growth factor [VEGF]‐A). All of the subjective and objective measurements collectively suggested that asthenopia was lessened for the 500 lx–4000 K condition. However, asthenopia was significantly worse for 300 lx–2700 K and 1000 lx–6500 K in terms of subjective symptoms and objective optical performance, respectively. Conclusions LED illuminance and CCT do have a significant effect on asthenopia during reading. 500 lx–4000 K lighting resulted in the lowest level of asthenopia. Conversely, low illuminance at low CCT (300 lx–2700 K) and high illuminance at high CCT (1000 lx–6500 K) promoted more severe asthenopia.