Mitochondria play a pivotal role in cellular function, not only as a major site of ATP production, but also by regulating energy expenditure, apoptosis signaling, and production of reactive oxygen species. Altered mitochondrial function is reported to be a key underlying mechanism of many pathological states and in the aging process. Functional measurements of intact mitochondria isolated from fresh tissue provides distinct information regarding the function of these organelles that complements conventional mitochondrial assays using previously frozen tissue as well as in vivo assessment using techniques such as magnetic resonance and near-infrared spectroscopy. This chapter describes the process by which mitochondria are isolated from small amounts of human skeletal muscle obtained by needle biopsy and two approaches used to assess mitochondrial oxidative capacity and other key components of mitochondrial physiology. We first describe a bioluminescent approach for measuring the rates of mitochondrial ATP production. Firefly luciferase catalyzes a light-emitting reaction whereby the substrate luciferin is oxidized in an ATP-dependent manner. A luminometer is used to quantify the light signal, which is proportional to ATP concentration. We also review a method involving polarographic measurement of oxygen consumption. Measurements of oxygen consumption, which previously required large amounts of tissue, are now feasible with very small amounts of sample obtained by needle biopsy due to recent advances in the field of high-resolution respirometry. We illustrate how careful attention to substrate combinations and inhibitors allows an abundance of unique functional information to be obtained from isolated mitochondria, including function at various energetic states, oxidative capacity with electron flow through distinct complexes, coupling of oxygen consumption to ATP production, and membrane integrity. These measurements, together with studies of mitochondrial DNA abundance, mRNA levels, protein expression, and synthesis rates of mitochondrial proteins provide insightful mechanistic information about mitochondria in a variety of tissue types.