Background To detect early cardiac deterioration, a simple and stable tool is needed. Measurement of time intervals in a simple 4-chamber view using color-coded tissue Doppler imaging is a relatively new approach to assess fetal cardiac function. The aim of this study was to evaluate the applicability of this modality and to construct reference ranges for cardiac time intervals. Methods We performed a prospective longitudinal cohort study in healthy fetuses. We used linear mixed models to construct age-adjusted reference ranges for shortening time (St) and lengthening time (Lt) in three cardiac regions: global heart and right and left ventricular wall. St and Lt were expressed as percentage of the cardiac cycle. Feasibility and intra-and interobserver variabilities were evaluated. We applied the technique to twin-twin transfusion syndrome (TTTS) recipients before laser therapy to test the diagnostic performance. Results A total of 251 recordings were obtained from 54 healthy singletons. St decreased and Lt increased with gestational age in all regions. We found a high feasibility (99.6%) and excellent intra-/interobserver variability for St (0.96/0.94) and Lt (0.99/0.96) of the global heart. Left and right ventricle performance parameters were good. In TTTS recipients, St was prolonged (p < 0.01) and Lt was shortened (p < 0.01) in all regions and the feasibility was excellent (96.6%). Conclusions The assessment of fetal cardiac function by measurement of cardiac time intervals is technically feasible with good reproducibility, even in difficult scanning circumstances such as TTTS. It is possible to discriminate between healthy and compromised fetuses with this technique.