Analysis of short-term (5-minute) heart rate variability (HRV) may provide useful information about autonomic nervous control of the cardiovascular system. The aim of the present study was to evaluate the association between the results of treadmill exercise testing (TET) and short-term (5-minute) HRV. Patients undertaking TET were anteriorly evaluated with short-term (5-minute) HRV over time (SDNN, RMSSD, NN50 count, pNN50) and frequency (LF, HF, total power) domains. Among 414 patients, 32 individuals (7.7%; 14 men) had nondiagnostic results. The nondiagnostic group had older age, higher body mass index, more hypertension, lower SDNN, lower LF, and higher HF than the negative group. After adjustment for potential confounders, SDNN (OR 0.94, 95% CI 0.91-0.97; P = 0.01), RMSSD (OR 0.96, 95% CI 0.93-0.99; P = 0.02), NN50 count (OR 0.98, 95% CI 0.97-1.00; P = 0.04) and LF (OR 0.96, 95% CI 0.96-0.99; P = 0.03) were negatively related to nondiagnostic results, and HF showed a positive effect (OR 1.04, 95% CI 1.01-1.05; P = 0.02). No HRV indices were significantly associated with positive results. Our study suggested that cardiac autonomic indices derived from short-term HRV recordings might predict nondiagnostic results of TET.