The objective of this study was to establish whether variations of amniotic fluid volume induced by second-trimester amniocentesis could be detected by serial measurements of amniotic fluid index. A total of 130 singleton pregnancies undergoing second-trimester amniocentesis for genetic indications were considered. Amniotic fluid index was measured at three different time intervals: 30-60 min before amniocentesis, immediately after the procedure, and 60 min after the procedure. Serial measurements were obtained either by a single operator (n = 55) or by the three independent operators (n = 75). Significantly lower amniotic fluid index values were demonstrated immediately after amniocentesis when compared with the pre-amniocentesis and subsequent measurements in the study design with both the single and multiple operators. No statistically significant changes were found between the first amniotic fluid index measurements and those obtained 1 h after amniocentesis. These results suggest that second-trimester amniocentesis induces a temporary decrease of amniotic fluid volume detectable by serial amniotic fluid index measurements, no longer evident 1 h after the procedure.