As the era of personalised medicine evolves, there is a growing interest in developing individually tailored cancer radiotherapy, where patients with intrinsic radiosensitivity are identified prior to commencing treatment to minimise severe adverse reactions. Patients without radiosensitivity could be given dose-intensified therapy to reduce locoregional failure and enhance survival. In a previous study of radiotherapy patients with known severe late radiation toxicity, we established a functional assay to predict individual radiosensitivity based on DNA repair analysis, which achieved 97% predictive power [1]. Here we further evaluated the clinical applicability of this assay in radiotherapy-treated non-small cell lung cancer (NSCLC) patients and aimed to detect changes in DNA repair in response to radiotherapy. The peripheral blood lymphocytes of 10 patients were ex vivo irradiated and DNA double-strand break (DSB) repair was analysed prior to, during and after radiotherapy, using the γ-H2AX assay [2]. A novel quantitative approach was employed to examine repair kinetics, as defined by an extensive range of multidimensional parameters, which were derived from the non-linear regression analysis of γ-H2AX foci. Radiation dose response and colocalisation efficiency with 53BP1 were also studied. While the repair rate and foci yield remained constant during and after radiotherapy, the unrepairable component of γ-H2AX foci decreased over the course of treatment. The largest magnitude of change occurred within 1 hour after the first fraction of radiotherapy. This enhanced DSB repair capacity could be attributed to radiation-induced adaptive responses. While the exact dose these lymphocytes received during radiotherapy and the generalisation of these results into normal and tumour tissues warrant further investigation, this study provides important new information to our predictive assay; that the parameters used to classify radiosensitivity vary across the time course of radiotherapy. Further modification and optimisation of this assay would increase its precision in characterising individual radiosensitivity, thereby allowing for individualisation of curative intent radiotherapy delivery.