Does tuning to a slightly lower reference pitch actually affect listeners, or is it a myth?

Separating those two questions is exactly right, and they have different answers.

Can people hear a difference? Yes, in the trivial sense that the whole piece is slightly lower — about a third of a semitone. Play any recording pitched down by that much and most listeners will describe it as warmer or heavier, and pitched up as brighter. That effect is real, well known, and has nothing to do with the specific numbers. Musicians transpose for exactly this reason.

Does 432 specifically have a special property? No. There is no credible evidence for it, and the claims made in its support do not survive checking — the numerology about resonance and natural frequencies is constructed after the fact, and the historical claims about it being an ancient standard are not supported by the record.

So: a real perceptual effect from being lower, and no evidence for anything special about that number rather than any other slightly lower number.

The historical context is worth knowing because it undercuts the "natural versus artificial" framing that the claim rests on.

Before standardisation, pitch varied enormously — by place, by period, by instrument type. Surviving organs and wind instruments span a range of well over a semitone. There was no single traditional pitch that 440 replaced; there was chaos, and orchestras had been drifting steadily upward for a long time because higher pitch sounds brighter and more brilliant.

The standard was agreed in the twentieth century precisely so that instruments and recordings could work together. Choosing 440 was a practical decision about a convention, not a departure from a natural order.

Which means the claim has the history backwards: there was no lost standard, only an absence of one.

There is a genuine musical argument for lower tuning that gets lost in the mystical framing, and it is worth rescuing.

For singers, a slightly lower pitch makes a demanding part easier. Repertoire written when performing pitch was lower is genuinely harder at the modern standard, and this is a real and much-discussed issue in some vocal music.

For period instruments, playing at a lower pitch is about historical practice and about what the instruments were built for — gut strings under less tension, different tone.

Both are legitimate reasons to tune lower and neither has anything to do with 432 in particular. Ensembles that do this typically use pitches associated with the repertoire's period rather than that number.

So if somebody wants a warmer sound or an easier vocal line, tuning down is a reasonable choice with reasons behind it. The reasons just are not the ones usually given.

One practical test anybody can run, and it is more persuasive than any argument: take a recording you know well and shift it by that amount both ways in any audio editor.

Listen without knowing which is which, ideally with somebody else operating it. Most people can hear a difference between the two. Almost nobody can reliably say which one is the "natural" one when they are not told, and the descriptions people offer flip depending on what they were told beforehand.

That asymmetry — a real audible difference, no consistent preference once expectation is removed — is the whole answer in one experiment.