Background: Microvascular dysfunction in the setting of ST-elevated myocardial infarction (STEMI) plays an important role in long-term poor clinical outcome. Coronary flow reserve (CFR) is a well-established physiological parameter to interrogate the coronary microcirculation. Together with hyperaemic average peak flow velocity, CFR constitutes the coronary flow capacity (CFC), a validated risk stratification tool in ischaemic heart disease with significant prognostic value. This mechanistic study aims to elucidate the time course of the microcirculation as reflected by alterations in microcirculatory physiological parameters in the acute phase and during follow-up in STEMI patients. Methods: We assessed CFR and CFC in the culprit and non-culprit vessel in consecutive STEMI patients at baseline (n = 98) and after one-week (n = 64) and six-month follow-up (n = 65). Results: A significant trend for culprit CFC in infarct size as determined by peak troponin T (p = 0.004), time to reperfusion (p = 0.038), the incidence of final Thrombolysis In Myocardial Infarction 3 flow (p = 0.019) and systolic retrograde flow (p = 0.043) was observed. Non-culprit CFC linear contrast analysis revealed a significant trend in C-reactive protein (p = 0.027), peak troponin T (p < 0.001) and heart rate (p = 0.049). CFC improved both in the culprit and the non-culprit vessel at one-week (both p < 0.001) and six-month follow-up (p = 0.0013 and p < 0.001) compared with baseline. Conclusion: This study demonstrates the importance of microcirculatory disturbances in the setting of STEMI, which is relevant for the interpretation of intracoronary diagnostic techniques which are influenced by both culprit and non-culprit vascular territories. Assessment of non-culprit vessel CFC in the setting of STEMI might improve risk stratification of these patients following coronary reperfusion of the culprit vessel.