Vergence and accommodation responses of human vision are very important factors when a 3D image is observed, and a vergence-accommodation conflict (VAC) causes perceptual distortion, visual discomfort, and fatigue for an observer. Theoretically, a hologram is expected to provide a 3D image without such a conflict. In this article, natural focusing was verified by human accommodation response (A-R) measurement during on-axis analog reflection Denisyuk hologram observation. The A-R of a group of participants were measured for a real marker and its Denisyuk hologram at various visualization distances using an Nvision K5001 autorefractor. The experimental results statistically confirmed the equivalence of the responses to the Denisyuk hologram and its real counterpart, as well as the absence of a VAC.