Sarcomas present as a heterogeneous disease entity with the hallmark characteristics of early onset, delayed diagnosis and poor prognosis. Recent technological advancements have underpinned the full exploitation of cancer-related genomic information, thereby enabling the identification of individuals with increased susceptibility to sarcoma and accordingly, the development of personalised treatment regimens. We aimed to determine if there was evidence for defective DNA damage and repair response in sarcoma patients with heterozygous mutations in ATM, BRIP1, BRCA1 and 2, PALB2, and components of the MRN complex (NBN and RAD51). Frozen peripheral blood lymphocytes (PBL) of 26 patients from the International Sarcoma Kindred Study (ISKS) cohort and their age- and sex-matched controls were thawed and then assessed for DNA damage response to ex vivo radiation using the γ-H2AX assay, a biomarker of radiation-induced double-strand breaks. DNA repair kinetics was evaluated by parameters derived from the non-linear regression analysis of the γ-H2AX response, including the fraction of unrepairable component (Q) and repair rate (R). Mutant-specific alterations, radiation dose-response and colocalisation efficiency with 53BP1 foci were also analysed. Overall, single-mutant patients had larger Q-values than controls (p = 0.020). Further analysis also indicated larger Q-values in individual single-mutant groups, with the largest difference shown between controls and BRCA1 group. The current study presented a novel approach for quantifying DNA damage and repair responses in PBL with high efficacy, precision and the potential for translation into clinical applications. While it did find evidence for DNA repair defects, the analysis was subjected to a considerable amount of variability due to the use of frozen lymphocytes as a sub-optimal choice of a cell model and limited knowledge about the radiosensitivity phenotype of patients. Analysis of fresh lymphocytes from a larger cohort and a better understanding of the functional consequences of each variant would improve future investigations.