In vivo, breathing movements including tidal and deep inspirations (DIs) exert a number of beneficial effects on respiratory system responsiveness in healthy humans, which are diminished or lost in asthma possibly as a result of reduced distension (strain) of airway smooth muscle (ASM).We used bronchial segments from pigs to assess airway responsiveness under static conditions and during simulated tidal volume oscillations with and without DI and to determine the roles of airway stiffness and ASM strain on responsiveness. Airway dilations during breathing were simulated by cycling the lumen volume of liquid filled segments. Volume oscillations (15 cycles/min) were set so that in relaxed airways they produced a ~5 to 10 cmH₂O increase in transmural pressure for tidal maneuvers, and ~5 to 30 cmH₂O for DIs. Acetylcholine dose-response-curves (10^-7 to 3 x 10^-3 M) were constructed under static and dynamic conditions and maximal response and sensitivity were determined. Airway stiffness was measured from tidal trough-to-peak pressure and volume cycles. ASM strain produced by DI was estimated from luminal volume, airway length and wall area. DIs produced substantial dilation (~40-50%) reflected by a decrease in both maximal response (P < 0.001) and sensitivity (P < 0.05). However, the magnitude of bronchodilation decreased significantly in proportion to airway stiffening caused by contractile activation and an associated reduction in ASM strain. Tidal oscillations in comparison had little effect on responsiveness. We conclude that DI regulates airway responsiveness at the airway level but this is limited by airway stiffness due to reduced ASM strain.