Mechanical dissection of single intact mammalian skeletal muscle fibers permits real-time measurement of intracellular properties and contractile function of living fibers. A major advantage of mechanical over enzymatic fiber dissociation is that single fibers can be isolated with their tendons remaining attached, which allows contractile forces (in the normal expected range of 300–450 kN/m²) to be measured during electrical stimulation. Furthermore, the sarcolemma of single fibers remains fully intact after mechanical dissection, and hence the living fibers can be studied with intact intracellular milieu and normal function and metabolic properties, as well as ionic control. Given that Ca²⁺ is the principal regulator of the contractile force, measurements of myoplasmic free [Ca²⁺] ([Ca²⁺]_i) can be used to further delineate the intrinsic mechanisms underlying changes in skeletal muscle function. [Ca²⁺]_i measurements are most commonly performed in intact single fibers using ratiometric fluorescent indicators such as indo-1 or fura-2. These Ca²⁺ indicators are introduced into the fiber by pressure injection or by using the membrane-permeable indo-1 AM, and [Ca²⁺]_i is measured by calculating a ratio of the fluorescence at specific wavelengths emitted for the Ca²⁺-free and Ca²⁺-bound forms of the dye. We describe here the procedures for mechanical dissection, and for force and [Ca²⁺]_i measurement in intact single fibers from mouse flexor digitorum brevis (FDB) muscle, which is the most commonly used muscle in studies using intact single fibers. This technique can also be used to isolate intact single fibers from various muscles and from various species. As an alternative to Ca²⁺ indicators, single fibers can also be loaded with fluorescent indicators to measure, for instance, reactive oxygen species, pH, and [Mg²⁺], or they can be injected with proteins to change functional properties. The entire protocol, from dissection to the start of an experiment on a single fiber, takes ∼3 h.