Nowadays a huge range of applications is deployed on Field Programmable Gate Arrays (FPGAs) devices. In some application domains, security requirements are arising and posing new design issues and challenges. Physically Unclonable Functions (PUFs) are emergent and promising solutions in providing some security mechanisms, such as key storing and generation, challenge/response provider, and protection of Intellectual Property (IP). Most widespread PUFs’ architectures are based on Ring Oscillators (ROs), which exploit comparisons between measured frequencies, obtained from selected RO pairs, in order to generate PUF responses. In this paper, we present a study of the frequencies characteristics, implementing ROs on the Xilinx Spartan 6 family, in order to statistically characterize the oscillations, evaluating the impact of some external uncontrolled parameters that can disturb and alter their original qualities.