I'd put in a repeat like this:

#!/usr/bin/perl

use strict;
use warnings;
use Tk;
use Tie::File;
use Tk::After;

#...

my $liste = $frame1->ScrlListBox(

#...

)->pack( -side => "right", -expand => 1, -fill => "both" );

$liste->repeat( 5000, \&fill_from_file );
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