Bundles of 3–5 fibres were activated at pCa 4.5 by a temperature jump from 1°C using a mechanical apparatus adapted to collect the X-ray diffraction pattern. Active isometric force was 176±30 kPa at 12°C (T_0,12), and 0.35 and 1.4 times T_0,12 at 4 and 20°C respectively. The lattice spacing d_1,1 at 4°C was 25.8nm in relaxed fibres and 24.6 nm during activation and decreased with increase in temperature with a slope that was larger in activated fibers. The M3 meridional reflection from the axial repeat of the myosin heads was sampled by X-ray interference between the two halves of the myosin filament. During activation the intensity ratio of the two main M3 peaks varied from 0.37±0.07 at 4°C to 0.45±0.12 at 20°C. M3 spacing (S_M3) was 14.480±0.006 nm in relaxed fibres and increased on activation. S_M3 increased with increasing temperature in active fibers, and the S_M3-force relation had a slope of 0.32±0.03%/T_0,12 and a zero-force intercept of 14.510±0.004 nm, 0.2% higher than S_M3 in relaxed fibers. These results show that (i) filament lattice spacing reduces with active force, with a slope, corrected for the temperature effect in relaxed fibers, of -2.9±1.4%/T_0,12, (ii) the increase in S_M3 on activation is much smaller than the 1.5% reported for intact frog fibres and (iii) the S_M3-force relation would correspond to a myosin filament compliance of ca 6.4nm/mm/T_0,12, similar to that in active frog fibres. Supported by MIUR and Ministero della Salute (Italy), MRC (UK), ESRF (EU).