BACKGROUND AND AIMS: Single-use electrocardiography (ECG) leads have been developed to reduce healthcare-associated infection. This study compared the validity and reliability of short-term heart rate variability (HRV) obtained from single-use disposable ECG leads. METHODS: Thirty healthy subjects (33 +/- 10 years; 9 females) underwent 5-min resting HRV assessments using disposable (single use) ECG cable and wire system (Kendall DL Cardinal Health) and a standard, reusable ECG leads (CardioExpress, Spacelabs Healthcare). RESULTS: Intraclass correlation coefficient (ICC) with 95% confidence interval (CI) between disposable and reusable ECG leads was for the time domain [R-R interval (ms); 0.99 (0.91, 1.00)], the root mean square of successive normal R-R interval differences (RMSSD) (ms); 0.91 (0.76, 0.96), the SD of normal-to-normal R-R intervals (SDNN) (ms); 0.91 (0.68, 0.97) and frequency domain [low-frequency (LF) normalized units (nu); 0.90 (0.79, 0.95), high frequency (HF) nu; 0.91 (0.80, 0.96), LF power (ms(2)); 0.89 (0.62, 0.96), HF power (ms(2)); 0.90 (0.72, 0.96)] variables. The mean difference and upper and lower limits of agreement between disposable and reusable leads for time- and frequency-domain variables were acceptable. Analysis of repeated measures using disposable leads demonstrated excellent reproducibility (ICC 95% CI) for R-R interval (ms); 0.93 (0.85, 0.97), RMSSD (ms); 0.93 (0.85, 0.97), SDNN (ms); 0.88 (0.75, 0.95), LF power (ms(2)); 0.87 (0.72, 0.94), and HF power (ms(2)); 0.88 (0.73, 0.94) with coefficient of variation ranging from 2.2% to 5% (p > 0.37 for all variables). CONCLUSION: Single-use Kendall DL ECG leads demonstrate a valid and reproducible tool for the assessment of HRV.