Background Heart Rate Variability (HRV) represents efferent vagus nerve activity which is suggested to be inversely related to fundamental mechanisms of tumorigenesis and to be a predictor of prognosis in various types of cancer. HRV is also believed to predict the occurrence and severity of post-operative complications. We aimed to determine the role of pre-operative HRV as a prognostic factor in overall and cancer free survival in patients with colorectal cancer. Methods Retrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January 2010 and December 2016, who underwent curative surgical treatment. HRV was measured as time-domain parameters (SDNN (Standard Deviation of NN-intervals) and RMSSD (Root Mean Square of Successive Differences)) based on pre-operative 10 second ECGs. Groups were created by baseline HRV: Low HRV (SDNN <20ms or RMSSD <19ms) and normal HRV (SDNN �20ms or RMSSD �19ms). Primary endpoints were overall and cancer free survival. Results A total of 428 patients were included in this study. HRV was not significantly associated with overall survival (SDNN <20ms vs SDNN �20ms:24.4% vs 22.8%, adjusted HR = 0.952 (0.607-1.493), p = 0.829; RMSSD <19ms vs RMSSD �19ms:27.0% vs 19.5%, adjusted HR = 1.321 (0.802-2.178), p = 0.274) or cancer recurrence (SDNN <20ms vs �20ms:20.1% vs 18.7%, adjusted HR = 0.976 (0.599-1.592), p = 0.924; RMSSD <19ms vs �19ms, 21.5% vs 16.9%, adjusted HR = 1.192 (0.706-2.011), p = 0.511). There was no