Ultra-short-term (UST) heart rate variability (HRV) metrics have increasingly been proposed as surrogates for short-term HRV metrics. However, the concurrent validity, within-day reliability, and between-day reliability of UST HRV have yet to be comprehensively documented. Thirty-six adults (18 males, age: 26 ± 5 yr, BMI: 24 ± 3 kg/m2) were recruited. Measures of HRV were quantified in a quiet-stance upright orthostatic position via three-lead electrocardiogram (ADInstruments, FE232 BioAmp). All short-term data recordings were 300 s in length and five UST time points (i.e., 30 s, 60 s, 120 s, 180 s, and 240 s) were extracted from the original 300-s recording. Bland-Altman plots with 95% limits of agreement, repeated measures ANOVA and two-tailed paired t tests demarcated differences between UST and short-term recordings. Linear regressions, coefficient of variation, intraclass correlation coefficients, and other tests examined the validity and reliability in both time- and frequency domains. No group differences were noted between all short-term and UST measures, for either time- (all P > 0.202) or frequency-domain metrics (all P > 0.086). A longer recording duration was associated with augmented validity and reliability, which was less impacted by confounding influences from physiological variables (e.g., respiration rate, carbon dioxide end-tidals, and blood pressure). Conclusively, heart rate, time-domain, and relative frequency-domain HRV metrics were acceptable with recordings greater or equal to 60 s, 240 s, and 300 s, respectively. Future studies employing UST HRV metrics should thoroughly understand the methodological requirements to obtain accurate results. Moreover, a conservative approach should be utilized regarding the minimum acceptable recording duration, which ensures valid/reliable HRV estimates are obtained.NEW & NOTEWORTHY A one size fits all methodological approach to quantify HRV metrics appears to be inappropriate, where study design considerations need to be conducted upon a variable-by-variable basis. The present results found 60 s (heart rate), 240 s (time-domain parameters), and 300 s (relative frequency-domain parameters) were required to obtain accurate and reproducible metrics. The lower validity/reliability of the ultra-short-term metrics was attributable to measurement error and/or confounding from extraneous physiological influences (i.e., respiratory and hemodynamic variables).