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The aims of this work are to characterize the paleoenvironmental evolution and the sedimentary fluvial responses to climate and river flow changes of the upstream reaches of the Sado estuary since the Mid-Late Holocene. By combining the previous information, we also intent to define the limit of the fluvial-estuarine boundary and maximum limit of tidal influence of the estuarine area during the last 4500 years. To accomplish these objectives a 10.5 m-long sediment core was collected at ca. 65 km upstream the Sado estuary inlet and studied using multiproxy analyses and radiocarbon dating. In addition, multi-proxy data from sediment cores and superficial samples collected downstream, in the alluvial plain and intertidal areas near the present-day fluvial-estuarine boundary, were combined and contributed to the interpretations. We concluded that: i) the organic matter is mostly sourced in terrestrial/freshwater environments even during periods with marine influence, as suggested by the occurrence of marine benthic diatoms between 4300 and 4200 cal BP; ii) the fluvial-estuarine boundary retreat during the Middle to Late Holocene transition (4350-4000 cal BP) reaching areas of the Sado river (Laxique), located ca. 15 km upstream of the present-day boundary; iii) modifications on the location of the fluvial-estuarine boundary are mostly derived from drier climatic conditions and consequent flow river discharges; iv) the aggradation and progradation of the alluvial plain started at ca. 4000 cal BP at Laxique (and at ca. 3240 cal BP at Arapouco); and v) the abrupt change in the sedimentation rates and sedimentation pattern before and after the beginning of the alluvial plain aggradation process (4000 cal BP at Laxique and 3240 cal BP at Arapouco) reflects essentially changes in the fluvial activity and the loss of accomodation space. [ABSTRACT FROM AUTHOR]