It is well established that hypoxia can influence tumor biology and physiology, gene expression, metastatic potential, treatment efficacy, and patient survival. Most human solid tumors have been shown to have some hypoxic regions, thus there is a strong motivation to understand the various causes of hypoxia. One key to understanding tumor hypoxia involves the study of oxygen transport to tumors, and the connection between hypoxia, tumor microvasculature, and the tumor microenvironment. Recent research has suggested that the causes of tumor hypoxia are much more complex than indicated by the classical paradigms ("chronic" and "acute" hypoxia), and several potential factors have been identified. Two such factors are temporal fluctuations in tissue pO2 and longitudinal gradients in oxygen transport. Research has shown the existence of low frequency (<2 cycles per minute) fluctuations in tumor pO2 without cessation of blood flow that can lead to transient hypoxia. In addition, longitudinal gradients in tumor pO2 along the arteriolar afferent direction have been documented in window chamber tumors. However, the causes of the pO2 temporal fluctuations and longitudinal gradients are not exactly known, and the clinical significance of these observations is not well understood. In this preliminary study, we demonstrate the potential of optical imaging measurements of hemoglobin saturation to add new information in these areas. Slow temporal fluctuations of hemoglobin saturation (HbSat) and gradients in the average HbSat were observed in some 4T1 mouse mammary carcinoma microvessels. With additional research, the mechanisms behind these phenomena and insights into their clinical significance may be revealed.