Co-seismic changes in groundwater regime are often observed after moderate to strong earthquakes. The 24 August 2016 Mw 6.0 extensional Amatrice earthquake, which was the first event of a long-lasting seismic sequence, including the 30 October 2016 Mw 6.5 Norcia event, triggered a significant discharge alteration to the Pescara di Arquata spring, located in the Umbria-Marche Apennines (Northern Apennines, Central Italy) and exploited for drinking purposes. During the first five months after the first mainshock, an extra flow of about 30% was recorded, while both water chemistry and temperature did not show significant changes. Thereafter, the spring discharge decreased significantly, and at the end of 2019 it was still lower than normal. The Standardized Precipitation Index (SPI) indicates that these low mean monthly discharge values are not related to particularly dry conditions. The increase in post-seismic depletion coefficients indicates that the aquifer empties faster than it did during the inter-seismic period. The observed transient increase and subsequent decrease of discharge are consistent with a transient, earthquake-related increase in hydraulic conductivity.