Ultra-short heart rate variability (HRV) metrics represent autonomic tone parameters derived using small epochs of interbeat interval data. These measures have risen in popularity with the advent of wearable devices that can capture interbeat interval data using electrocardiography (ECG) or photoplethysmography. Autonomic neuropathy in diabetes mellitus (DM) is well established, wherein 5-min HRV is conventionally used. Ultra-short measures have the potential to serve as markers of reduced autonomic tone in this patient population. Data of patients with Type I and Type II DM who had presented to our laboratory for autonomic neuropathy assessment were chosen for analysis. One-minute and 2-min epochs were chosen from 5 min of ECG data using standard software. Time domain, frequency domain, and nonlinear measures were computed from 1 to 2 min epochs, and reliability was compared with measures derived from 5-min HRV using intraclass correlation coefficients (ICCs). Data of 131 subjects (79 males, 52 females; mean age = 53.3 ± 12.16 years) were analyzed. All ultra-short HRV measures derived from 1 min to 2 min data showed good to excellent reliability (median ICC values ranging from 0.83 to 0.94) when compared with 5-min metrics. The notable exception was very low frequency (VLF) power, which showed poor reliability (median ICC = 0.43). Ultra-short HRV metrics derived from 1 to 2 min epochs of ECG data can be reliably used as predictors of autonomic tone in patients with DM. VLF power is poorly reproducible in these small epochs, probably due to variability in respiratory rates. Our findings have implications for ultra-short HRV estimation using short epochs of ECG data.