In situ stress measurements were performed during high frequency pulse electrodeposition of nanotwinned Cu thin films. Periodic stress changes during pulse-on and pulse-off periods were observed. The stress profile showed an abrupt increase in tensile stress to about 400 MPa during the pulse-on period and a stress relaxation during the pulse-off period. First-principles calculations predict that a complete relaxation of the tensile stress allows the formation of nanotwins separated by 28 nm or more. This is in good agreement with the results obtained from microstructural analysis of the Cu films fabricated during in situ stress measurements.