| Experimental procedures.

(A) Ultrasonic stimulation protocol for Experiments I & II. In Experiment III a duty cycle of 10% was used. In Experiment IV a stimulus duration of 400 ms was used. (B) TUS-TMS clamp (DOI: 10.5281/zenodo.6517599). (C) Experimental timing. Detailed experimental timing for each experiment is reported in Supplementary Fig. 3.

| Ultrasonic stimulation parameters.

f = fundamental frequency, depth = TPO focus setting for distance of free-water full-width half-maximum from transducer exit plane, PD = pulse duration, PRF = pulse repetition frequency, DC = duty cycle, PTD = pulse train duration, Isppa = spatial-peak pulse-average intensity in free-water, P = pressure, MItc = transcranial derated mechanical index. The ramp shape for all experiments was rectangular. For estimated intracranial indices for Experiments I & II see Supplementary Figure 2.

| Experimental conditions.

On-target TUS of the left-hemispheric hand motor area (Exp. I-III), active control TUS of the right-hemispheric face motor area (Exp. I-II), sound-only sham (Exp. I-III), and inactive control TUS of the white matter ventromedial to the hand motor area (Exp. IV). Conditions involving TUS were presented both with and without auditory masking stimuli.

| Non-specific motor inhibitory effects of TUS.

A significant suppression of MEP amplitude relative to baseline (gray) was observed for on-target TUS (green), but also for stimulation of a control region (cyan), and presentation of a sound alone (sound-sham; blue) indicating a spatially non-specific and sound-driven effect on motor cortical excitability. There were no significant differences between on-target and control conditions. Bar plots depict condition means, error bars represent standard errors, clouds indicate the distribution over participants, and points indicate individual participants. Square-root corrected MEP amplitudes are depicted for Experiments I, II, and IV, and Relative MEP amplitude is depicted for Experiment III (see Methods). *p < 0.05, **p < 0.01, ***p < 0.001.

| No significant dose-response effects of TUS.

(A) Acoustic (top) and thermal (bottom) simulations for a single subject in Experiment II. The acoustic simulation depicts estimated pulse-average intensity (Ipa) above a 0.15 W/cm2 lower bound, with the dotted line indicating the full-width half-maximum of the pressure. The thermal simulation depicts maximum estimated temperature rise. (B) On-target TUS MEP amplitude as a percentage of active control MEP amplitude against simulated intracranial intensities at the two applied free-water intensities: 6.35 W/cm2 (top) and 19.06 W/cm2 (bottom). The shaded area represents the 95% CI, points represent individual participants. No significant intracranial dose-response relationship was observed. (C) There is no significant effect of free-water stimulation intensity on MEP amplitude. Values are expressed as a percentage of baseline MEP amplitude (square root corrected). Remaining conventions are as in Fig. 3. (D) Temporal autocorrelation, operationalized as the slope of the linear regression between trial t and its preceding baseline trial t-1, differed significantly as a function of stimulation site and intensity for masked trials. Individual points represent the differential autocorrelation compared to the active control site. Autocorrelation was not modulated during baseline or sound-only sham, but was significantly higher for on-target TUS at 6.35 W/cm2, and significantly lower for on-target TUS at 19.06 W/cm2 compared to active control TUS. *p < 0.05, **p < 0.01, ***p < 0.001.

| No effects of time-locked masking.

There were no significant effects of time-locked masking, indicating that audible differences between stimulation sites did not obscure or explain the absence of direct neuromodulation. Conventions are as in Figs. 3 and 4C.

| Sound-driven effects on corticospinal excitability.

(A) Longer (auditory) stimulus durations resulted in lower MEP amplitudes, regardless of TUS administration, indicating a sound-duration-dependency of motor inhibitory outcomes (Exp. I). (B) A significant effect of auditory stimulus duration was also observed in Experiment III. (C) The pitch of auditory stimuli also affected MEPs, where lower amplitudes were observed following a 1 kHz tone. There was no effect of TUS. Conventions are as in Figs. 3 and 4C.

| Sound-driven effects on corticospinal excitability.

Less MEP attenuation was measured during continuous masking, particularly for lower stimulation intensities (i.e., auditory confound volumes), pointing towards a role of TUS audibility in MEP attenuation.

| Auditory cueing of TMS.

There was a significant reduction in MEP amplitude when participants were first presented with a 500 ms stimulus (initial trials) in Experiment I (left) and Experiment II (right), following by a stabilization of MEP amplitude during the rest of the experiment (following trials), indicating a learning process by which TUS acts as a cue signaling the onset of TMS. The solid line depicts the loess regression fit, and the shaded area represents the 95% confidence interval.

| Contribution diagram.

This figure depicts the involvement of each author using the CRediT taxonomy (Brand et al., 2015) and categorizes their contributions according to three levels represented by color: ‘none’ (gray), ‘substantial contribution’ (light green), ‘leading contribution’ (dark green).