Background: Familial ST-segment Depression Syndrome (Fam-STD) is a novel inherited arrhythmia disease associated with atrial fibrillation, ventricular arrhythmias, and sudden cardiac death. The first aim of the current project is to examine the electrical activity pathway of the heart in Fam-STD patients compared to healthy controls. The second aim is to complete an in-depth analysis of the ST-segment in Fam-STD patients. Methods: Retrospective CineECG analysis of data from 27 patients with Fam-STD and 83 age- and gender-matched controls. ECG features, mean Temporal-Spatial Isochrones (mTSI), which describes the average location of all cardiac electric activity, and ST-segment 10ms intervals per lead, were analyzed. Results: Twenty-seven Fam-STD patients (26% males, mean age 51.6+-6.2 years) and 83 controls (28% males, 51.4+-1.0 years) were included. Significant differences were found in heart rate (63.0+-4.5 & 71.3+-3.2 (p < 0.001)) and QTc (395.4+-7.7 & 413.7+-6.0 (p < 0.001)) between Fam-STD and controls, respectively. The most prominent differences were found in mTSI directions, described as direction of activation given in the direction of X = posterior-anterior, Y = right-left and Z = base-apex. End-QRS to ST (direction-Z) (0.8+-0.10 & -0.4+-0.2 (p < 0.001)), ST-peak (direction-Z) (0.5+-0.2 & -0.8+-0.1 (p < 0.001)), and in the STT (direction-Z) (0.3+-0.2 & -0.9+-0.0 (p < 0.001)) between Fam-STD and controls, respectively. The most pronounced ST-segment deviations in Fam-STD group, were found at 70-79 and 80-89ms post J-point, in lead II (-0.167 & -0.167mV), V4 (-0.171 & -0.169mV) and V5 (-0.178 & -0.177mV), respectively. Conclusions: This analysis found pronounced differences in mTSI direction-Z, in time intervals near the ST segment, which indicates the electrical current, in Fam-STD patients, moves towards the base during the ST segment, opposite to healthy subjects. A detailed ST-analysis showed pronounced ST-deviations, in Fam-STD patients, in lead II, V4 and V5, highest in the 70-89ms interval.