The purpose of this investigation was to cross-validate the ithlete heart rate variability smart phone application with an electrocardiograph for determining ultra-short-term root mean square of successive R-R intervals. The root mean square of successive R-R intervals was simultaneously determined via electrocardiograph and ithlete at rest in twenty five healthy participants. There were no significant differences between the electrocardiograph and ithlete derived root mean square of successive R-R interval values (p > 0.05) and the correlation was near perfect (r = 0.99, p < 0.001). In addition, the ithlete revealed a Standard Error of the Estimate of 1.47 and Bland Altman plot showed that the limits of agreement ranged from 2.57 below to 2.63 above the constant error of -0.03. In conclusion, the ithlete appeared to provide a suitably accurate measure of root mean square of successive R-R intervals when compared to the electrocardiograph measures obtained in the laboratory within the current sample of healthy adult participants. The current study lays groundwork for future research determining the efficacy of ithlete for reflecting athletic training status over a chronic conditioning period.