Natural killer (NK) cells are critical in the first-line defense against viral infections. Chronic HIV-1 infection leads to a perturbation in the NK cell compartment, yet the kinetics of this deregulation and the functional consequences are unclear. Here, we characterized changes in the NK cell compartment longitudinally by multiparameter flow cytometry, starting in acute HIV-1 infection. Acute HIV-1 infection was associated with elevated NK cell numbers, with an expansion of CD3^negCD56^dimCD16^pos NK cells and an early depletion of CD3^negCD56^brightCD16^neg NK cells. Ongoing viral replication resulted in a depletion of CD3^negCD56^dimCD16^pos NK cells with a paralleled increase in functionally anergic CD3^negCD56^negCD16^pos NK cells, accompanied by reduced functional activity, as measured by CD107a expression and cytokine secretion. Taken together, these studies demonstrate a sequential impairment of NK cell function with persistent viral replication resulting from a progressive deregulation of NK cell subsets with distinct functional properties.